CA2141679C - New derivatives of neuraminic acid - Google Patents

New derivatives of neuraminic acid Download PDF

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CA2141679C
CA2141679C CA002141679A CA2141679A CA2141679C CA 2141679 C CA2141679 C CA 2141679C CA 002141679 A CA002141679 A CA 002141679A CA 2141679 A CA2141679 A CA 2141679A CA 2141679 C CA2141679 C CA 2141679C
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ethylglycoside
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Aurelio Romeo
Gunter Kirschner
Hari Manev
Martino Trimarco
Gino Toffano
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    • 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
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    • 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
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    • 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
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Abstract

Provided are new derivatives of neuraminic acid of formula (I), where Ac represents an aryl residue of an aliphatic, araliphatic aromatic, alicyclic, or heterocyclic carboxylic acid, including carboxylic amides, their 2-hydrocarbyl-glycosides, and their peracylated derivatives at the hydroxy groups of both these series of amides. These compounds are therapeutically useful in providing a protective effect against the neurotoxicity induced by excitatory amino acids, and can therefore be used in therapies of the central nervous system following cerebral degenerations or lesions, metabolic dysfunctions, aging, and toxic-infective and chronic neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases.

Description

WO 94/03469 2141 ~~ 7 9 PCT/US93/07307 NEW DERIVATIVES OF :L~1EUR.AMINIC ACID
BACKGROUND OF 'THE INVENTION
Field of the Invention The present invention rE~lates to new derivatives of neuraminic acid, especially carboxylic amides of the following formula, N OH
W ~ ~ ~ (I>
H/ ~~_ WO
Ar+N
where Ac represents an acyl residue of a carboxylic acid of the aliphatic, araliphatic, aromatic, alicyclic or heterocyclic series, comprie;ing 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 :.~rvous syste,.,, such as those following cerebral degener,ations 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 carboxylic amides and their derivatives of formula I according the present invention are new.
Description of Related Art In the literature, there is a description of the non-substituted amide of N-acetyl-neuraminic acid, prepared as an intermediate in the synthesis of tetrazolyl-2-decarboxy-N-acetyl-neuraminic acid (see Ann. 1986, 2104-11).
In an article published in Hoppe Seyler's Physiol. Chemie, 1983, 364 (109) 1411-17, there is a description of the amides obtainable through the reaction of the benzylketoside of N-acetyl-neuraminic acid with L-glycine, L-glutamic acid, and L-phenylalanine, followed by the elimination of the benzyl group through catalytic hydrogenation; no pharmacological action is described for these derivatives.
SUMMARY OF THE INVENTION
In addition to providing new derivatives of neuraminic acid, 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.
Further scope of the applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and w~ 3 ~ l 41679 scope of the invention will become apparent to those skilled in the art from this detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of 'ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
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-, -S02-, or phenylene groups in the carbon atom chain of the hydrocarbylic residue. The halogen atoms are preferentially fluorine, bromine, or chlorine.
Esterified hydroxylic or mercapto groups can derive f rom w 2141679 WO 94/03469 I'Cf/US93/07307 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 hydrocarbylic residues.
Finally, they can derive from hydrocarbylsulfonic acids .
Etherified hydroxy or mercapto groups,' or esterified carboxylic or sulfonic groups, preferentially derive from alcohols of the aliphatic series having no more than 8 carbon atoms, or from the araliphatic series with only one benzene ring and an alkylene of 1 or 2 carbon atoms. The hydrocarbylic groups which can substitute the aminic groups preferentially derive from these alcohols; the amino groups can also be in the form of quaternary ammonium salts, e.g., tetraalkyl groups, e.g., tetrabutylammonium.
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 -O-, -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 O
-O-S-~-~ -O-p ~ , or -O_p~0 \OH \O _ and in those of esters with hydrocarbylsulfonic acids (e.g., p-toluenesulfonic or methanesulfonic acid), by -O-S02.
The hydrocarbylic reside Ac, as already stated, can be blocked by sulfoxide or sulfdriyl residues. In amides, converted carboxylic or sulfonic groups preferentially derive from lower aliphatic amines with WO 94/03469 ~ ~~ 416 7 9 , p~/US93/07307 not more than 4 carbon atom:, 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 5 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. Of particular interest are the following acids: butyric, valeric, particularly normal valeric and isovaleric, trimethylacetic (pivalic acid), caproic, isocaproic, enantic, caprylic, pelargonic, capric, undecilic, di-tert-butyl-acetic, 2-propyl-valeric (valproic acid), lauric, tridecilic, myristic, pentadecilic, palmitic, margaric, stearic, arachic, behenic, and lignoceric.
Among substituted aliphatic acids, 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.
The Ac group of formula I can also derive from natural or synthetic peptides preferentially having not more than 12 amino acid:, selected from naturally occurring amino acids, e.g., those aforesaid.
Of particular interest according to the present invention are those in which Ac is an acyl residue belonging to peptides of the thymus gland. Acids, from which derive an Ac group of araliphatic nature, are e.g., phenylacetic, cinnamic, phenylpropionic or atropic acid. Among aromatic acids, benzoic acid and its methylatPd homologues, salicylic acid, anthranilic acid, trimethoxybenzoic acid, pht:halic or terephthalic acid, o,o'-dicarbonic acid, chlorobenzoic acid, vanillic acid, and veriatric or piperonilic acid must be mentioned.

WO 94/03469 ~ ~ ~ ~ ~ ~ PCT/US93/073~'1 Among Ac acyl groups belonging to acids of the alicyclic series, there 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.
If 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, dihydrolysergic, 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, -CONH2), 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-S02- group. These amines have no more than 24 carbon atoms.
The functions which can eventually substitute the carbon atom chain of the amide or the amine are preferentially those mentioned for the Ac group of formula I. Aliphatic amines can have an open, saturated, unsaturated, linear, branched or cyclic chain. Of particular interest are alkyl- and dialkylamines having from 1 to 12 carbon atoms, such as, for example, methylamine, ethylamine, propylamine, hexylamine, diethylamine, dimethylamine, diisopropylamine, WO 94/03469 2 1 . ~ ~ 9 PCT/US93/07307 dihexylamine and alkylenylamines having from 3 to 6 cyclic carbon atoms, wherein the rings are substituted or non-substituted, preferentially between one and three C1-14 alkyl groups, e.c~., methyl groups, like pyrrolidine, piperidine, and azepine.
The hydrocarbylic chains can also be blocked with heteroatoms such as, for e:xample, -O-, -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. Of particular interest are the following: aliphatic diamines, like ethylenediamine, trimethylenediamine, tetramethylenediamine, penta- and hexamethylenediamine, piperazine and its N-alkyl o:r C-alkyl derivatives having a C1-4 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. Moreover, morphol.ine 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.
Of particular interest also are some derivatives of amines having a large number of carbon atoms of an aliphatic nature, and which .are related to phospholipids or sphingolipids or similar derivatives.
According to the present invention, 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: phosphatidylethano7_amine, phosphatidylserine, sphingosine, dihydrosphingosine, psychosine, dihydropsychosine, phosphorylcholine-sphingosine, WO 94/03469 '~, ~ ~ ~ ~~ ~ PCT/US93/073m s 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 C1-4 lower aliphatic hydrocarbylic groups such as, for example, aniline, ~anthranilic acid, 1-amino-4-sulfonic acid, and benzylamide. In the aforesaid aliphatic amines, in one or more positions of the hydrocarbylic chain, 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.
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 C1-4 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 °

~ WO 94/03469 PCT/US93/07307 2;141679 ~3 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. Among 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. Among alcohols of the araliphat:ic 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. Among alcohols of the alicyclic series having more rings, steroid alcohols such as, for example, those of the pregnane group, like cortic:osteroids, and among these methylprednisolone, must be mentioned. Among alcohols of the heterocyclic series there must be mentioned tetrahydrofuranol, tetrahyd:ropyranol, furfuryl alcohol and pyridylcarbinol.
In 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 c~~n therefore derive from hydroxyacids like lactic a<:id, from amino acids like glycine, or from dibasic acids like succinic, malonic or y-WO 94/03469 ~ ~ 416 7 9 PCT/US93/07307 1. 0 malefic acid. Among 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, 5- p-aminobenzoic, salicylic and phthalic acid.
The new compounds according to present invention can eventually be transforme=d 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 respect to this equivalence between salts and amides in free form, it is obvious that what will be de=scribed 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 of the Present Invention Defined 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-neuraminic acid, phthaloyl-neuraminic acid, chlorobenzoyl-neuraminic acid, vanilloyl-neuraminic ~~ JO 94/U3469 1416 7 9 . , I'CI'/US93/U73U7 acid, cyclopenthane- and cycl.ohexane-carbonyl-neuraminic acid, N-nicotinyl-neuraminic acid, N-isonicotinyl-neuraminic acid, lisergyl-neuraminic acid, 2-bromo-lisergyl-neuraminic acid, 1.-methyl-lisergyl-neuraminic acid, theophillineacetyl-neuraminic acid, and their 2-glycosides derived from one of the following alcohols:
methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, butyl alcohol, i;sobutyl alcohol, tertbutyl alcohol, ethyleneglycol, propyleneglycol, benzyl alcohol, methylprednisolone, tetrah~rdrofuranol, tetrahydropyranol, fu:rfuryl alcohol, and pyridylcarbinol.
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 <~cids having an acyl group deriving from one of the following acids: aminobutyric, methionine, lysine, aspart is acid, glutamic acid, proline, tryptophan, ~or from an acyl residue deriving from a peptide present ~i.n the thymus, and their 2-glycosides deriving from one of the following alcohols : methyl alcohol , etlhyl alcohol , propyl alcohol , isopropyl alcohol, butyl <~lcohol, isobutyl alcohol, tertbutyl alcohol, ethyleneglycol, propyleneglycol, benzyl alcohol, methyl-predr~isolone, tetrahydrofuranol, tetrahydropyranol, fu:rfuryl alcohol, and pyridylcarbinol.
Another group of interesting compounds according to the present invention is foamed by the.amides deriving from pyrrolidine, piperidine, azepine, ethylenediamine, trimethylenediamine, hexamethylenediamine, piperazine or N-methyl or N-ethyl-piperazine, aminoethanol, aminopropanol, mercapto~ethylamine, morpholine, tiomorpholine, or peptides like those present in the thymus, and from phosphatidylethanolamine, phosphatidylserine, sp:hingosine, psychosine, ':.

WO 94/03469 ~ PC1"/US93/07307 dihydropsychosine, sphingosylphosphorylcholine, dihydrosphingosylphosphoryl~~holine, or from the phytosphingosine of one of the following N-acyl-neuraminic acids: N-palmitoyl-neuraminic acid, N-stearoyl-neuraminic acid, N-acetyl-neuraminic acid, N-propionyl-neuraminic acid, N-pivaloyl-neuraminic acid, N-valeroyl-neuraminic acid, I~&-caproyl-neuraminic acid, N-lauroyl-neuraminic acid, N,--'succinyl-neuraminic acid, phenylacetyl-neuraminic acid, phthaloyl-neuraminic acid, chlorobenzoyl-neuraminic acid, N-nicotinyl-neuraminic acid, N-isonicotinyl-neuraminic acid, lisergyl-neuraminic acid, 2-bromo-lisergyl-neuraminic acid, 1-methyl-lisergyl-neuraminic acid, theophillineacetyl-neuraminic acid, and their 2.-glycosides derived from one of the following alcoho7.s: methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, tertbutyl alcohol, ethyleneglycol, propylene:glycol, benzyl alcohol, methylprednisolone,tetrhydrofuranol,tetrahydropyranol, furfuryl alcohol, and pyrid.ylcarbinol.
Other derivatives- are: those peracylated at the hydroxy groups of N-aryl-neuraminic acid, and particularly peracetates, perpropionates, perbutyrates, pervalerianates, perpiva7.ates, persuccinates and perbenzoates.
Synthesis of the compounds of the present invention The present invention also comprises processes for the preparation of the new amides of N-acyl-neuraminic acids, their 2-hydrocarbylc~lycosides, and their salts.
These processes are a:Lready known, and consist of the stepwise introduction of the amine function and eventually of the 2-g:lycosidic group into an N-acyl-neuraminic acid, and eventually of acyl groups -into the hydroxy groups and the final formation of their salts.

WO 94/03469 2 ~ 4 16 7 9 PCT/US93/07307 In a preferred embodirnent, 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 dE~rivatives, 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 soalt, 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 ~~onsists 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 Dowe~s~ type or a similar resin. As an example, it is possible to use the condensation method in presence of carbodiimides, e.g., dicyclohexylcarbodiimide,benzylisopropylcarbodiimide or benzylethylcarbodiimide, in the presence of 1-hydroxybenzo-triazol, or the condensation in the presence of N,N'carbonyl-di.imidazol. Starting from the aforesaid acidic derivatives, like esters or Trademark WO 94/03469 ~ (~ PCT/US93/07307 :L 4 halogenides, e.g., bromides or chlorides, 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.
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-O-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.
If acylation of the amine group of neuraminic acid is performed at the end of the procedure, e.g., after amide or glycoside for;nation, 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 transformation of the final compounds into their salts, as well as the interconversion of the salts, is performed in a known manner, as for example when intermediate salts are prepared for their purification.
The aforesaid procedure according to the present invention also comprises all variations in which the procedure is stopped at evE=ry step, or in which the starting compound is an intermediate, or in which the starting compounds are prepared in situ.

WO 94/03469 2 : s "~ PCT/US93/07307 The synthesis of the compounds of the present invention is illustrated by the following examples.
Examx~le 1 Butilamide of N-acetylneuraminic acid 5 3.23, g (10 ~ mM) of N-acetylneuraminic acid methylester, prepared according to Kuhn et al., Chem Ber. 99,611 (1966), were solubilized in 50 ml of anhydrous methyl alcohol; 3.66 g (50 mM) of 2-butylamine were added. The mixture was stirred for 5 hours at 40 10 °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 butilamide of N-acetyl-neura~minic acid were gathered and 15 evaporated under vacuum. T'he residue was crystallized from 50 ml of n-propyl alcohol. Yield: 85%.
Rf - 0.25, chloro:form/methyl alcohol/water, 110:40:6.
.Examt~le 2 Q-2-O-ethylalycoside of butilamide of N-acetylneuraminic acid 3.65 g (10 mM) of t:he (3-2-O-ethylglycoside of N-acetylneuraminic acid ethyl ester, prepared according to Kuhn et al., Chem Ber. 99, 611 (1966), were solubilized in 80 ml of anhydrous methyl alcohol; 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, 80:20:2. The fractions containing the ~3-2-O-ethylglycoside of the butilamide of N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was crystallized from 50 ml of n-propyl alcohol and 100 ml of ethyl ether. Yield: 70%.

Rf = 0.37, chloroform/methyl alcohol/water, 110:40:6;
0.19, chloroform/methyl alcohol/2.5N NH40H, 80:20:2.
Example 3 a-2-O-ethvlctlvcoside of the benzylamide of N-acetylneuraminic acid 3.65 g (10 mM) of the ~3-2-O-ethylglycoside of N-acetylneuraminic acid ethyl ester, prepared according to Kuhn et al., Chem Ber. 99, 611 (1966), were solubilized in 50 ml of anhydrous methyl alcohol; 5.36 g (50 mM) of benzylamine 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, 80:20:2. The fractions containing the ~i-2-O-ethylglycoside of the benzylamide of N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was crystallized from 50 ml of isopropyl alcohol. Yield:
65%.
Rf = 0.50, chloroform/methyl alcohol/water, 110:40:6;
0.16, chloroform/methyl alcohol/2.5N NH40H, 80:20:2.
Example 4 a-2-O-ethvlalycoside of the dimethylaminopropylamide of N-acetylneuraminic acid 3.65 g (10 mM) of the (3-2-O-ethylglycoside of N-acetylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methyl alcoh~7.; 10.2 a (lOn 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 WO 94/03469 _ 214 I ~~ ~ 9 PCT/US93/07307 1'7 chloride/methyl alcohol/2.5N NH40H, 55:45:10. The fractions containing the ~i-2-O-ethylglycoside of dimethylaminopropylamide of N-acetyl-neuraminic acid were gathered and evaporateol under vacuum. The residue was dissolved in 50 ml of water. Yield: 75%.
Rf = 0.19, chloroform,/methyl alcohol/2.5N NH40H, 40:60:15.
Exam~~le 5 Q-2-O-ethvlctlvcoside of the dimethylaminopropylamide of N-acetylneuraminic acid (mal.eic acid salt) 3.65 g (10 mM) of tine /3-2-O-ethylglycoside of N-acetylneuraminic 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/2.5N NH40H, 55:45:10. The fractions containing the ~i-2-O-ethylglycoside of the dimethylaminopropylamide of: N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of wager, a stoichiometric amount of malefic acid was added, and the material was lyophilized. Yield: 75%.
Rf = 0.19, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.
Exammle 6 a-2-O-ethylalycoside of the dimethylamide of N-acetvlneuraminic acid 3.65 g (10 mM) of t:he ~i-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 WO 94/03469 ~ ~ ~ ~ ~ ~ (~

chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 80:20:2. The fractions containing the,-2-O-ethylglycoside of the dimethylamide of N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was crystallized from 30 ml of methanol and 150 ml of ethylrether. Yield: 80%.
Rf = 0.38, chloroform/methyl alcohol/water, 110:40:6.
Example 7 a-2-O-ethvlctlvcoside of the dimethylaminopropylamide of N-palmitoylneuraminic acid 5.62 g (10 mM) of the ~3-2-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 solvent a mixture of methylene chloride/methyl alcohol/water, 110:40:6. The fractions containing the /3--2-O-ethylglycoside of the dimethylaminopropylamide of N-palmitoyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of water and lyophilized. Yield:
70 %.
Rf = 0.12, chloroform/methyl alcohol/2.5N NH40H, 80:20:2.
Example 8 -2-O-ethvlctlycoside of the dimethvlaminopropvlamide of N-palmitoylneuraminic acid (malefic acid salt) 5.62 g (10 mM) of the ~i-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 2.141 ~'~9 ....

chromatography, using as solventt a mixture of methylene chloride/methyl alcohol/wat:er, 110:40:6. The fractions containing the ~i-2-O-ethylglycoside of the dimethylaminopropylamide of N-palmitoyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water, a stoichiometric amount of malefic acid was added, and the material was lyophilized. Yield: 70%.
Rf = 0.12, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.
Example 9 a-2-O-ethvlglvcoside of the dimethylaminopropylamide of N-nalmitoylneuraminic acid 5.48 g (10 mM) of t:he a-2-O-ethylglycoside of N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methy7_ 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 a-2-O-ethylglycoside of the dimethylaminopropylamide of N-palmitoyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of water and lyophilized. Yield:
70%.
Rf = 0.40, chloroform/methyl alcohol/0.3% CaCl2, 60:40:9.
Examp:L a 10 a-2-O-ethylalycoside of the dimethvlaminopropvlamide of N-palmitoylneuraminic acid (male~~ acid salt?
5.48 g (10 mM) of the a-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 solvent a mixture of methylene chloride/methyl alcohol/water, 110:40:6. The fractions 5 containing the a-2-O-ethylglycoside of the dimethylaminopropylamide of N.=~palmitoyl-neuraminic acid were gathered and evaporated'under vacuum. The residue was dissolved in 50 ml of water, a stoichiometric amount of malefic acid was added, and the material was 10 lyophilized. Yield: 70%.
Rf = 0.40, chloroform/methyl alcohol/0.3% CaCl2, 60:40:9.
Example 11 Q-2-O-ethylglvcoside of the dimethylamide of 15 N-palmitoylneuraminic acid 5.56 g (10 mM) of the ~i-2-O-ethylglycoside of N-palmitoyl-neuraminic acid, sodium salt, were dissolved in 50 ml of pyridine, and 2.3 g (20 mM) of pyridinium chloride and 4.12 g (20 mM) of N,N'dicyclohexyl-20 carbodiimide were added. The mixture was stirred for 2 hours at 25 °C. 4.5 g (100 mM) of dimethylamine 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/water, 80:10:1. The fractions containing the ~i-2-O-ethylglycoside of the dimethylamide of N-palmitoyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of hexane. Yield:
90%.
Rf = 0.69, chloroform/methyl alcohol/water, 110:40:6.

1 ~~.16 7 9 p~/US93/07307 2 :L
Example 12 a-2-O-ethylcrlycoside of the dimethylamide of N-palmitoylneuraminic acid 5.48 g (10 mM) of the a-2-O-ethylglycoside of N-palmitoylneuraminic 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 so7Lution 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. The fractions containing the a-2-O-ethylglycoside of the dimethylamide of N-palmitoyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of hexane. Yield: 90%.
Rf = 0.69, chloroform/methyl alcohol/water, 110:40:6.
Examnl e: 13 a-2-O-ethylctl~rcoside ~ of the butyl amide of N-acetvlneuraminic acid 3.65 g (10 mM) of the a-2-O-ethylglycoside of N-acetylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methyl alcohol; 3.66 g (50 mM) of butylamine were added. 'The mixture was stirred overnight at 25 °C. The solution was evaporated under vacuum and the residue way; purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 80:20:2. The fractions containing the a-2-O-ethylglycoside of the butylamide of N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was crystallized from 50 ml of methanol and 300 ml of ethyl ether. Yield: 75%.

WO 94/03469 PCT/US93/07zn;
~14~~79 Rf - 0.55, chloroform/methyl alcohol/water, 110:40:6;
Rf - 0.53, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.
Example .14 a-2-O-ethylQlycoside of the dimethylaminopropvlamide of N-acetylneuraminic acid 3.65 g (10 mM) of the a-2-O-ethylglycoside of N-acetylneuraminic 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/2.5N NH40H, 55:45:10. The fractions containing the a-2-O-ethylglycoside of the dimethylaminopropylamide of N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water and lyophilized. Yield:
700.
Rf = 0.21, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.
Example 15 a-2-O-ethvlglycoside of thedimethvlaminopropvlamide of N-acetylneuraminic acid (malefic acid salt) 3.65 g (10 mM) of the a-2-O-ethylglycoside of N-acetylneuraminic 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/2.5N NH40H, 55:45:10.
The fractions containing the a-2-O-ethylglycoside of the dimethylaminopropylamide of N-acetyl-neuraminic acid 21416.79 were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water, a stoichiometric amount of malefic acid was added, and the material was lyophilized. Yield: 70$.
Rf - 0.21, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.
Example 16 a-2-O-ethylalycoside of N-ace~tvlneuraminic acid amide with L-alanine-D-iso9~lutamine 3.65 g (10 mM) of the a-2-O-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 1N HCl, 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 hydro-chloride (prepared according to Le Francier (Bull. Soc. Chim.
Biol., Vol. 49, No. 10 (196;7)) and Kusumoto (Bulletin of the Chemical Society of Japan, Vol. 49(2), pp 533-539 (1976)) 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 acide, 4:1:1, and hydrogenated in an H2 current in the presence of BaS04-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 a-2-0-ethylglycoside of N-acetyl-neuraminic acid amide with L-alanine-D-iso-glutamine were gathered and evaporated under vacuum. The residue was *Trademark r dissolved in 200 ml of wager and lyophilized. Yield:
600.
Rf - 0.56, chloroform~/methyl alcohol/2.5N NH40H, 60:35:8;
Rf = 0.18, chloroform/methyl alcohol/0.3% CaCl2, 60:40:9.
Exam~> 1 a 17 Peracetylated a-2-O-ethylglycoside of N-palmitovl-neuraminic acid amide with L-alanine-D-isoglutamine 5.56 g (10 mM) of the a-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. 18 ml of anhydrous pyridine and 10.:? g of acetic anhydride were added and stirring was cond'.ucted 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 methy~lene 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 1N HC1 and twice with 50 ml of water. The organic phases were anhydrified with anhydrous sodium sulfate, gathered, and evaporated under vacuum. The residue was di:~solved 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 (2o mM) of Trademark - ~ WO 94/03469 ~ ~41 G 7 9 pCT/U593/07307 c. 5 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 H2 current in the presence of BaS04-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 a-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 200 ml of water and lyophilized. Yield:
55%.
Rf = 0.54, chloroform/methanol, 90:10.
Examp:l a 18 2-O-ethvlalvcoside of N-~~cetvlneuraminic acid amide with L-alanine-D-isog~lutamine 3.59 g (10 mM) of the ~i-2-O-ethylglycoside of N-acetylneuraminic acid, sodium salt, prepared according to Eschenfelder and Brossmer, Hoppe Seyler's Z. Physiol.
Chem. 360, 1253 (1979), 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 canducted 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, ~~ JVO 94/03469 2 ~ 416 7 9 ~'~/US93/07307 :26 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 1N HC1 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. 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 terbutylat:e 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, the residue was dissolved in 30 ml of water, and 10 ml of 1N NaOH were added. 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 (3-2-O-ethylglycoside of N-acetyl-neuraminic acid amide with ~ L-alanine-D-isoglutamine were gathered and Trademark WO 94/03469 2 ~ 416 7 9 PCT/US93/07307 evaporated under vacuum. The residue was dissolved in 50 ml of water and lyophilized. Yield: 600.
Rf = 0.12, chloroform/methyl alcohol/2.5N NH40H, 60:35:8;
0.10, chloroform/methyl alcohol/0.3o CaCl2, 60:40:9.
Example 19 I3-2-O-ethylcxlycoside of N-palmitovlneuraminic acid amide with L-alanine-D-isoctlutam~ine 5.56 g (10 mM) of the ~i-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. 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 anh~rdrous 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 1N HC1 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 Trademark WO 94/U34G9 2 ~ ~ ~ 6 7 9 I'Cf/US93/U7307 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. 5 ml of anhydrous Dower 50x8 resin, H+ form, wera added. The solution was filtered and evaporated under vacuum. The residue was dissolved in 30 ml of water, and 10 ml of 1N NaOH were added. 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 (3-2-O-ethylglycoside~ of N-palmitoyl-neuraminic acid amide with L-alanine-D-isoglutamine were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of a mixture of water/dioxane, 4:1, and lyophilized. Yield: 60%.
Rf = 0.12, chloroform/methyl alcohol/2.5N NH40H, 110:40:6.
0.57, chloroform/methyl alcohol/0.3o CaCl2, 60:40:9.
Example 20 a-2-O-ethylg~coside of N~-acetvlneuraminic acid amide with arainine 3.65 g (10 mM) of the a-2-O-ethylglycoside of 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 *Trademark 'VO 94/03469 21416 l 9 PCT/US93/07307 25 °C for 30 minutes, neutralized with 1N HCl, and eluted with water from a column containing 30 ml of Dowex* 50x8 resin, pyrid_Lnium 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. 4.64 g (15 mM) of N-nitro-L-arginine ben.zyl ester (prepared according to Bonnaud, Bull Chim. Farcn. 121, 1982) 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 H2 current in the presence of BaS04-supported palladium. After filtration, the solution was evaporated, and the residue was purified by silica gel chromatography, using as aolvent a mixture of methylene chloride/methyl alcohol/water, 60:40:9. The fractions containing the a-2-O-ethylglycoside of N-acetyl-neuraminic acid. amide with arginine were gathered and evaporated Lender vacuum. The residue was dissolved in 50 ml of water and lyophilized. Yield: 50%.
Rf = 0.13, chlorofo~rm/methyl alcohol/0.3% CaCl2, 60:40:9.
Example 21 -2-O-ethylqlycoside of N-acetvlneuraminic acid amide with arainine 3.59 g (10 mM) of the ~i-2-O-ethylglycoside of N-acetylneuraminic 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 iEor 24 hours at 35 °C. The Trademark r~WO94/03469 ~~ 6~~ ~ PCT/US93/07307 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, 5 anhydrified with anhydr~ous~ .. sodium sulfate, and evaporated under vacuum. ,fihe obtained residue was solubilized in 50 ml of anh~idrous N, N' -dimethylformamide at 25 °C; 2.64 g (20 mM) of sodium thiophenate were added and the mixture wa;~ stirred for 4 hours. The 10 solution was evaporated under high vacuum. The residue was extracted three times with 200 ml of ethyl acetate, washed with 100 ml of cold 1N HC1 and twice with 50 ml of water. The organic phases were anhydrified with anhydrous sodium sulfate, gathered, and evaporated under 15 vacuum. The residue was solubilized in 100 ml of anhydrous pyridine; 7..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. After 15 20 minutes, the temperature was raised to 25 °C and the mixture was stirred for 5 hours. 4.64 g (15 mM) of N-nitro-L-arginine benzyl ester hydrochloride (prepared according to Bonnaud, Bull. Chim. Farm. 121, 1982) were added at 25 °C. The mixtux-e was stirred overnight and 25 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. 5 ml of anhydrous Dowex 50x8 resin, H+ form, were added. The solution was filtered 30 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 H2 current in the presence of BaSO~-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/wat=er, 110:40:6. The fractions VO 94/03469 1 ~I 1 ~ 7 PCf/US93/07307 :31 containing the /3-<'?-O-ethylglycoside of N-palmitoyl-neuraminic acid amide with arginine were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water and lyophilized. Yield: 50%.
Rf - 0.10, chloroform/methyl alcohol/0.3% CaCl2, 60:40:9.
Example 22 !3-2-O-ethylglycoside of N-palmitovlneuraminic acid amide with arcrinine 5.56 g (10 mM) of the (3-2-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 meth~~lene chloride. The organic phases were washed twice with 50 ml of vuater 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 1N HC1 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 resin, pyridinium form. The eluate was lyophilized and then solubil.ized in 100 ml of anhydrous pyridine; 1.15 g (10 mM) of N-hydroxysuccinimide, 4.13 g (20 mM) of N,N'-dicyclohe:xylcarbodiimide and 11.6 g Trademark WO 94/03469 2 ~ 't ~1 b 7 9 P~T/US93/07307 :32 (10 mM) of pyridinium chloride were added at -10 °C.
After 15 minutes, the temperature was raised to 25 °C
and the mixture was stirred. for 5 hours. 4.64 g (15 mM) of N-nitro-L-arginine benzyl ester hydrochloride - 5 (prepared according to Bonnaud, Bull. Chim. Farm. 121, 1982) 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. 5 ml of anhydrous Dowex*50x8 resin, H+ form, were added. The solution was filtered and evaporated under vacuum. The residue was dissolved in, 100 ml of a mixture of n-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H2 current in the presence of BaS04-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/1.5M acetic acid, 110:40:6. The fractions containing the Q-2=O-ethylglycoside of N-palmitoyl-neuraminic ac~.d amide with arginine were gathered and evaporated under vacuum. The residue was crystallized from a mixture of 100 ml of methanol and 300 ml of ethyl ether. Yield: 60%.
Rf = 0.12, chloroformfmethyl alcohol/H20, 110:40:6.
Example 23 a-2-O-ethylglycoside of N-palmitoyl-neuraminic acid amide with arainine 5.56 g (10 mM) of the a-2-O-ethylglycoside of N-palmitoyl-neuraminic acid, sodium salt, were solubilized in 40 ml of wal:er and eluted with water from a column containing 30 ml of 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'-dicyclohexylcarbodiirnide, and 11.6 g (10 mM) of Trademark WO 94/03469 21416 7 9 p~/US93/07307 pyridinium chloride were added at -10 °C. After 15 minutes, the temperature was raised to 25 °C and the mixture was stirred for 5 hours. 4.64 g (15 mM) of N-vitro-L-arginine benzyl ester hydrochloride (prepared according to Bonnaud, Bull. Chim. Farm. 121, 1982) were added at 25 °C. The mixture was stirred overnight and then evaporated under vacuum. The residue was dissolved in 100 ml of a mixture of n-butanol/water/acetic acid, 4:1:1, and hydrogenated in an H2 current in the presence of BaS04-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/1.5M acetic acid, 110:40:6. The fractions containing the a-2-O-ethylglycoside of N-palmitoyl-neuraminic acid amide with arginine were gathered and evaporated under vacuum. The residue was crystallized from a mixture of 40 ml of methanol and 150 ml. of ethyl ether. Yield: 700.
Rf = 0.14, chloroform/methyl alcohol/H20, 110:40:6;
0.32, chlcroform,~methyl alcohol/2.5N NH40H, 60:35:8.
Example 24 Dimethylamide of N-palmitovlneuraminic acid 5.34 g (10 mM) of t:he a-2-O-ethylglycoside of N-palmitoyl-neuraminic acid were suspended in a mixture of 100 ml of O.1M H2S04/e~~hanol, 4:1, at 60 °C, and stirred for 16 hours. The product was extracted once 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, 4.5 g (100 mM) of dimethylamine were added at 25 °C and the solution wa~> stirred for 24 hours. The mixture was evaporated, and the residue was purified by WO 94/03469 PCT/US93/0730°' silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/water, 80:20:2. The fractions containing the dimethylamide of N-acetyl-neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of a mixture of water/dioxane, 4:.1, and lyophilized. Yield:
75%.
Rf = 0.63, chloroform/methyl alcohol/H20, 110:40:6.
Example 25 a-2-ethylctlycoside morpholino-propylamide of N-palmitoylneuraminic acid 5.62 g (10 mM) of ~i-2-ethylglycoside N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 14.4 g (100 mM) of N-(3-aminopropyl)-morpholine 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, 80:10:1. The fractions containing the compound were gathered and evaporated under vacuum. The residue was dissolved in 100 ml of water and lyophilized. Yield: 85%.
Rf = 0.30, chloroform/methyl alcohol/H20, 80:20:2;
0.62, chloroform/methyl alcohol/2.5N NH40H, 110:40:6.
Example 26 Q-2-ethylglycoside morpholino-propylamide of N-.palmitovlneuraminic acid (malefic acid salt) 5.56 g (10 mM) of ~i-2-O-ethylglycoside N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of 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. 14.4 g (100 mM) of N-(3-aminopropyl)-morpholine were added. The mixture was ' WO 94/03469 21 g~ 1 ~ 7 9 PCT/US93/07307 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/wate;r, 80:10:1. The fractions 5 containing the(3-2-ethylglycoside morpholino-propylamide of N-palmitoylneuraminic acid were gathered and evaporated under vacuum. Th~~ residue was dissolved in 100 ml of water. After adding a stoichiometric amount of malefic acid, the mixture was lyophilized. Yield: 85%.
10 Rf = 0.30, chloroform/methyl alcohol/H20, 80:20:2;
0.62, chloroform/methyl alcohol/2.5N NH40H, 110:40:6.
Example 27 Dimethylaminopropylamide of h1-palmitoyl-neuraminic acid 15 5.34 g (10 mM) of tree a-2-O-ethylglycoside of N-palmitoyl-neuraminic acid were suspended in a mixture of 100 ml of O.1M H2S04/etlzanol, 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 20 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 25 stirred for 2 hours. To the' filtered solution 10.2 g (100 mM) of dimethyl-aminopropylamine were added at 25 C and the solution was stirred for 24 hours. The mixture was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of 30 methylene chloride/methyl alcohol/2.5N NH40H, 60:35:8.
The fractions containing the compound were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of a mixture of water/dioaane, 1:2, and lyophilized.
Yield: 70%.
35 Rf = 0.21, chloroform/methyl alcohol/0.3% CaCl2, 60:40:9.

WO 94/03469 ~ ~ ~'~ ~~ 9 PCT/US93/0730', Examt~le 28 Dimethvlaminopropvlamide of N-palmitoyl-neuraminic acid (malefic acid salt) 5.34 g (10 mM) of the a-2-O-ethylglycoside of N-palmitoyl-neuraminic acid were suspended in a mixture of 100 ml of O.1M H2S04/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 (100 mM) of dimethyl-aminopropylamine were added at 25 °C, and the solution was stirred for 24 hours. The mixture was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/2.5N NH40H, 60:35:8.
The fractions containing the compound were gathered and evaporated under vacuum,. The residue was dissolved in 60 ml of a mixture of water/dioxane, 1:2. A stoichiometric amount of malefic acid was added, and the mixture was lyophilized. Yield: 70%.
Rf = 0.21, chloroform/methyl alcohol/0.3% CaCl2, 60:40:9.
Example 29 a-2-O-ethylctlvcoside butylamide of N-palmitoylneuraminic acid 5.62 g (10 mM) of ,Q-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/wat:er, 80:10:1. The fractions containing the a-2-O-ethylglycoside butylamide of N-palmitoylneuraminic acid ~Nere gathered and evaporated under vacuum. The residue was dissolved in 60 ml of dioxane and lyophilized. Yield: 70%.
Rf = 0.71, chloroform/methyl alcohol, 80:20;
0.60, chloroform~'methyl alcohol/2.5N NH40H, 80:20:2.
Examn:Le 3 0 a-2-O-ethvlqlycoside dimethylaminoethylamide of N-palmitovlneuraminic acid 5.62 g (10 mM) of ~i-2-O-ethylglycoside N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 4.4 g (50 mM) of 2-dimethylaminoethylamine were added. The mixture was stirred overnight at 40 °C. The solution was evaporated under vacuum and the residuf: was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/2..5N NH40H, 80:20:2. The fractions containing the (3-2-O-ethylglycoside dimethylaminoethylamide of N-palmitoylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of water and lyophilized. Yield:
80%.
Rf = 0.11, chloroform/methyl alcohol, 70:30;
0.43, chloroform%methyl alcohol/2.5N NH40H, 110:40:6.
Examp7_e 31 a-2-O-et~lalycoside dinnethvlaminoethylamide of N-palmitoylneuraminic acid (malefic acid salt) 5.56 g (10 mM) of ~i-2-O-ethylglycoside 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-he:xylcarbodiimide 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 NH40H, 80:20:2. The fractions containing the ~3-2-O-ethylglycoside dimethylaminoethylamide of N-palmitoylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of water. A stoichiometric amount of malefic acid was added, and the mixture was lyophilized. Yield: 80%.
Rf = 0.11, chloroform/methyl alcohol, 70:30;
0.43, chloroform/methyl alcohol/2.5N NH40H, 110:40:6.
Example 32 a-2-O-ethylctlycoside dimethylaminoprogylamide of N-dichloroacetylneuraminic acid 4.34 g (10 mM) of ,Q-2-O-ethylglycoside N-dichloro-acetylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 10.2 g (100 mM) of dimethylamino-propylamine 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, 40:60:15. The fractions containing the ~i-2-O-ethylglycoside dimethyl-aminopropylamide of N-dichloroacetylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of water and lyophilized.
Yield: 65%.
Rf = 0.37, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.

~VO 94/03469 2 ~ 416 l 9 PCT/US93/07307 Example 33 ~-2-O-ethylglycoside dimethylaminopropylamide of N-dichloroacetylneuraminic acid (malefic acid salt) 4.34 g (10 mM) of the ~i-2-O-ethylglycoside of_ N-dichloro-acetylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 10.2 g (100 mM) of dimethylamino-propylamine 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, 40:60:15. The fractions containing the ~i-2-O-ethylglycoside dimethyl-aminopropylamide of N-dichloroacetylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 60 ml of water. A
stoichiometric amount of m<~leic acid was added, and the mixture was lyophilized. '.Field: 65%.
Rf = 0.37, chloroform/methyl alcohol/2.5N NH40H, 40:60:15.
Examx~le 34 ~(i-2-O-ethylg~lycoside di~methylaminopro,~ylamide of neuraminic acid 3.23 g (10 mM) of the ~i-2-O-ethylglycoside of neuraminic acid ethyl ester, prepared according to Schauer and Buscher, Biochim. Biophys. Acta 338, 369 (1974), were solubilized in 50 ml of anhydrous methanol;
10.2 g (100 mM) of dimethylamino-propylamine were added.
The mixture was stirred overnight at 40 °C. The solution was evaporated under vacuum and the residue was purified by reverse phase chromatography, using as support Lichroprep* RP 18 (Merck, Darmstadt, Germany) and as eluant a mixture of methyl alcohol/water, 1:1. The fractions containing the ~i-2-O-ethylglycoside dimethyl-aminopropylamide of neuraminic acid were gathered and evaporated under vacuum. The residue was *Trademark WO 94/03469 . PCT/US93/07307 ~1~.~~'~ 9 40 dissolved in 50 ml of water and lyophilized. Yield:
60%.
Rf = 0.1, chloroform/methyl alcohol/0.3% CaCl2, 55:45:10.
Example 35 Q-2-O-ethylctlvcoside dimethylamide of N-dichloroacetylneuraminic acid 4.34 g (10 mM) of the /3-2-O-ethylglycoside of N-dichloro-acetylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 4.5 g (100 mM) of dimethylamine were added. The mixture was stirred overnight at 40 °C. The so:Lution 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. The fractions containing the ~i-2-O-ethylc~lycoside dimethyl-amide of N-dichloroacetylneuraminic acid were gathered and evaporated under vacuum. The residue was crystallized from 60 ml of methanol a.nd 300 ml of ethyl ether.
Yield: 60%. .
Rf = 0.44, chloroform/methyl alcohol/2.5N NH40H, 110:40:6.
Example 36 a-2-O-ethylcLlycoside ethanolamide of N-palmitoyl-neuraminic acid 5.48 g (10 mM) of the a-2-O-ethylglycoside of N-palmitoylneuraminic acid ethyl ester were solubilized in 50 ml of anhydrous methanol; 6.11 g (100 mM) of ethanolamine were added. The mixture was stirred overnight at 35 °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, 90:10. The fractions containing the a-2-O-ethylglycoside et.hanolamide of N-palmitoyl-neuraminic acid were gathered and evaporated under WO 94/03469 2 ~ 't ~16 7 9 ~'CT/US93/07307 ~41 vacuum. The residue was dissolved in 60 ml of dioxane/water, 2:1, and lyophilized. Yield: 85%.
Rf = 0.66, chloroform/methyl alcohol/H20, 110:40:6.
Example 37 Q-2-O-ethylc~lycoside dimethylamide of neuraminic acid 3.23 g (10 mM) of the (3-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized in 50 ml of water and 11 ml (10 mM) of 1M NaOH were added. The solution was maintained at :Z5 °C for 30 minutes. 2 m1 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 ai. 25 °C. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/2.5N NH40H, 110:40:6. The fractions containing the ~i-2-O-ethylglycoside dimethylamide of. neuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of water and lyophilized.
Yield: 55%.
Rf = 0.30, chloroform/rnethyl alcohol/H20, 55:45:10.
Examp:l a 3 8 Q-2-O-ethvlQlycoside dimethvlamide of N-caorylneuraminic acid 3.23 g (10 mM) of the Q-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in 100 ml anhydrous methanol. and 50 ml of anhydrous methylene chloride. 3.25 g (40 mM) of N,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 3.44 g (20 mM) of capric acid were added, and the mixture was stirred overnight at 5 °C.
Trademark WO 94/U34G9 1'Cf/US93/U73U7 2141 bl9 After filtration, it was evaporated. The residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1M 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. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by siluca gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the (3-2-O-ethylglycoside dimeth;ylamide of N-caprylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of n-hexane. Yield: 65%.
Rf = 0.48, chloroform/methyl alcohol/HZO, 80:10:1.
Examx~le 39 Q-2-O-ethvlalvcoside dimethylamide of N-ca~rvlovl-neuraminic acid 3.23 g (10 mM) of t:he ,Q-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in 100 ml anhydrous methanol. and 50 ml of anhydrous methylene chloride. 8.25 g (40 mM) of N,N'-dicyclohexylcarbodiimi~de, 2.0 g (20 mM) of triethylamine, and 2.88 g (20 mM) of caprylic acid were added and the mixture was stirred overnight at 5 °C.
After filtration, it was evaporated. The residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mMj of 1M 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 ~v Trademark 21~~b79 solubilized in 50 ml of anhydrous pyridine. 2.3 g (20 mM) of pyridinium chloride and 4.12 g (20 mM) of N,N'-dicyclohexylcarbodiim~.de were added. The mixture was stirred for 2 hours at 25 °C. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the ~i-2-O-ethylglycoside dimethylamide of N-capryloylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of n-hexane.
Yield: 65%.
Rf = 0.61, chloroform/methyl alcohol/H20, 80:20:2.
Examt~le 40 Q-2-O-ethylglycoside dimethylamide of N-olevlneuraminic acid 3.23 g (10 mM). of t:he ~i-2-O-ethylglycoside of neuraminic acid ethylester 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 5.65 g (20 mM) of oleic acid were added, and the mixture was stirred overnight at 5 °C.
After filtration, it was evaporated. The residue was solubilized in 100 ml of et:hanol/water, 1:1, and 11 ml (10 mM) of 1M 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 ~) of N,N'-dicyclohexylcarbodiimi~de were added. The mixture was stirred for 2 hours at: 25 °C. 4.5 g (100 mM) of diethylamine were added and. the reaction was conducted Trademark ~~ WO 94/03469 2 r 416 7 9 E'Cr/US93/07307 overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture o:E methylene chloride/methyl alcohol, 9:1. The fractions containing the R-2-O-ethylglycoside dimetlzylamide of N-oleylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of tert-butanol and lyophilized. Yield: 50%.
Rf = 0.42, chloroform/methyl alcohol/H20, 80;20:2.
Examx~ 1 a 41 a-2-O-ethylglycoside dimethylamide of N-valprovl neuraminic acid 3.23 g (10 mM) of the ~i-2-O-ethylglycoside of neuraminic acid ethylester were solubilized at 5 °C in 100 ml of anhydrous methanol and 50 ml of anhydrous methylene chloride. E..25 g (40 mM) of N,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 2.88 g (20 mM) of valproic acid were added and the mixture was stirred overnight at 5 °C.
After filtration, it was .evaporated. The residue was solubilized in 100 ml of ethanol/water, l:l, and 11 ml (10 mM) of 1M NaOH weres 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. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the (3-2-O-ethylglycoside dimethylamide of N-valproylneuraminic acid were gathered and evaporated *Trademark y0 94/03469 '~ 6 ~ PCT/US93/07307 under vacuum. The residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of n-hexane.
Yield: 500.
Rf = 0.60, chloroform/methyl alcohol/H20, 80:20:2.
5 Example 42 (3-2-O-ethylglycoside dimethylamide of N-phenylacetvl neuraminic acid 3.23 g (10 mM) of t:he ~i-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in 10 100 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 2.72 g 1;20 mM) of phenylacetic acid were added, and the mixturE: was stirred overnight at 5 15 °C. After filtration, it way; evaporated. The residue was solubilized in 100 ml of et:hanol/water, 1:1, and 11 ml (10 mM) of 1M 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 20 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. 4.5 g (100 mM) of 25 diethylamine were added, and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the 30 (3-2-O-ethylglycoside dimethylamide of N-phenylacetylneuraminic acid were gathered and evaporated under vacuum. The: residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of n-hexane. Yield: 65%.
35 ,Rf = 0.43, chloroform/methyl alcohol/H20, 80:20:2.
Trademark m WO 94/03469 2 1 4 1~6 7 9 PCf/US93/07307 Examx~le 43 L3-2-O-ethvlglvcoside dimetllylamide of N-miristovl neuraminic acid 3.23 g (10 mM) of the ~i-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. x;.25 g (40 mM) of N,N'-dicyclohexylcarbodiimi.de, 2.0 g (20 mM) of triethylamine, and 4.57 g (20 mM) of miristic acid were added, and the mixture was stirred overnight at 5 °C.
After filtration, it was evaporated. The residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1M 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. 4.5 g (100 mM) of diethylamine were added and. the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the ,Q-2-O-ethylglycoside dimethylamide of N-miristoylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated in 20 volumes of n-hexane.
Yield: 60%.
Rf = 0.56, chloroform/methyl alcohol/H20, 80:20:2.
Example 44 13-2-O-ethvlcrlycoside dimethvlamide of N-laurovl-neuraminic acid 3.23 g (10 mM) of tlae Q-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized at 5 °C in Trademark ,CVO 94/03469 214 ~ 6 7 9 P~/US93/07307 100 ml of anhydrous methanol and 50 ml of anhydrous methylene chloride. 8,.25 g (40 mM) of N,N'-dicyclohexylcarbodiimi.de, 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.
After filtration, it was evaporated. The residue was solubilized in 100 ml of et=hanol/water, 1:1, and 11 ml (10 mM) of 1M 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. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the Q-2-O-ethylglycoside dimethylamide of N-lauroylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated i.n 20 volumes of n-hexane.
Yield: 60%.
Rf = 0.54, chloroform/methyl alcohol/H20, 80:20:2.
Examp.l a 4 5 Q-2-O-ethylcr~coside dimeth~,rlamide of N-nicotinovl-neuraminic acid 3.23 g (10 mM) of the a-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.
Trademark CVO 94/03469 2 ~~ 416 7 9 ~ PCT/US93/07307 After filtration, it was evaporated. The residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1M NaOH were added. The solution was maintained at 25 °C for 3C) minutes. 2 ml of anhydrous Dower 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. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the ~i-2-O-ethylglycoside dimethylamide of N-nicotinoylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of methanol and precipitated in 20 volumes of tert-butyl-ether. Yield: 50%.
Rf = 0.27, chloroform/methyl alcohol/H20, 80:20:2.
Examp:L a 4 6 ~i-2-O-ethvlcLlycoside dimethvlamide of N-rrimethoxy benzovlneuraminic acid 3.23 g (10 mM) of the Q-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 4.24 g (20 mM) of trimethoxybenzoic acid were added, and the mixture was stirred overnight at 5 °C. After filtration, it was evaporated. The residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1M NaOH were added. The solution was maintained at 25 °C for 30 minutes. 2 ml of anhydrous Dower 50x8 resin,, H+ form, were added. The Trademark WO 94/03469 ~ ~ ~ ~ ~ ~,~ PCT/US93/07307 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. 4.5 g (100 mM) of diethylamine were added and the reaction was conducted overnight at 25 °C. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the ~i-2-O-ethylglycoside dimethylamide of N-trimethoxbenzoylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of methanol and precipitated in 20 volumes of tert-butyl-ether. Yield: 60%.
Rf = 0.52, chloroform/methyl alcohol/H20, 80:20:2.
Examp7Le 47 a-2-O-ethylql_ycoside pvrrol:idylamide of N-palmitoyl-neuraminic acid 5.56 g (10 mM) of the a-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. The solution was evaporated, and the residue was purified by silica gel chromatography, using as solvent a mixture of methylene chloride/methyl alcohol, 9:1. The fractions containing the (3-2-O-ethylglycoside pyrrolidylamide of N-palmitoylneuraminic acid were gathered and evaporated under vacuum. The residue was dissolved in 50 ml of acetone and precipitated i.n 20 volumes of n-hexane.
Yield: 90%.
Rf = 0.67, chloroform/methyl alcohol/H20, 80:20:2.

-- wo 94/o3a69 21 41 b 7 9 Examx> 1 a 4 8 Q-2-O-ethylglycoside of N-palmitoylneuraminic acid ethyl ester 3.23 g (10 mM) of the ~i-2-O-ethylglycoside of 5 neuraminic acid ethyl ester were solubilized at 5 °C in 100 ml of anhydrous methanol and 50 ml of anhydrous methylene chloride. F3.25 g (40 mM) of N,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 5.12 g (20 mM) of palmitic acid were 10 added, and the mixture wasc stirred overnight at 5 °C.
After filtration, it was evaporated. The residue was solubilized in 100 ml of ethanol/water, 1:1, and 11 ml (10 mM) of 1M NaOH were added. The solution was maintained at 25 °C for 30 minutes. 2 ml of anhydrous 15 Dower 50x8 resin, H+ form, were added. The filtered solution was evaporated under vacuum. 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 20 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 (3-2-O-ethylglycoside of N-palmitoylneuraminic acid 25 were gathered and evaporated under vacuum. The residue was crystallized from 100 ml of tert-butyl-ether. Yield:
85%.
Rf = 0.44, methylene ~~hloride/methanol, 90:10.
3 0 Examj~ 1 a 4 9 (3-2-O-ethvlcrlycoside of 1~-palmito~rlneuraminic acid 5.65 g (10 mM) of the ~i-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized in 100 ml of ethanol/water, 1:1. 11 ml (10 mM) of 1M NaOH were 35 added. The solution was maintained at 25 °C for 30 minutes. 2 ml of anhydrou;~ Dower 50x8 resin, H+ form, Trademark "" WO 94/03469 21416 7 9 PCT/US93/07307 were added. The filtered solution was evaporated under vacuum. Yield: 95%.
Rf = 0.14, methylene chloride/methyl alcohol/H20, 110:10:6.
Example 50 a-2-O-ethylcrlvcoside of N-t~almitovl-neuraminic acid methyl ester 3.37 g (10 mM) of the a-2-O-ethylglycoside of neuraminic acid methyl ester were solubilized in 30 ml of 1M NaOH at 80 °C, and maintained under stirring overnight. The solution was passed through a column containing 200 ml of Bio-Rex*70 H+ weakly basic resin, and then dessicated under vacuum. The residue was redissolved with 50 ml of anhydrous methanol. 4.13 g (20 mM) of N,N'-dicyclohexylcarbodiimide and 2.3 g of pyridinium chloride were added, and the mixture was stirred for 1 hour. After filtration, it was evaporated.
The residue was solubilized at 5 °C in 100 ml of anhydrous methanol and 50 ml of anhydrous methylene c h 1 o r i d a . 8 . 2 5 g ( 4 0 m M ) o f N,N'-dicyclohexylcarbodiimide, 2.0 g (20 mM) of triethylamine, and 5.12 g (20 mM) of palmitic acid were added, and the mixture wa;~ stirred overnight at 5 °C.
After filtration, it was evaporated. 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 chlorofor~cn; 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 a-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 = 0.65, methylene chloride/methanol, 90:10.
Trademark "''WO 94/03469 2 1 .416 l 9 PCT/US93/07307 Example 51 a-2-O-ethylqlycoside of N-palmitoylneuraminic acid sodium salt 5.47 g (10 mM) of the a-2-O-ethylglycoside of -neuraminic acid methyl ester were solubilized in 100 ml of methanol/water, 1:1. 11 ml (10 mM) of 1M NaOH were added. The solution was maintained at 25 °C for 30 minutes. 2 ml of anhydrous Dower 50x8 resin, H+ form, were added. The filtered solution was evaporated under vacuum. Yield: 95%.
Rf - 0.25, chloroform/methyl alcohol/H20, 110:10:6.
Example 52 13-2-O-ethylcLlycoside of N-dichloroacetvlneuraminic acid ethyl ester 3.23 g (10 mM) of the (3-2-O-ethylglycoside of neuraminic acid ethyl ester were solubilized in 50 ml of anhydrous pyridine at 25 °C; 14.3 g (100 mM) of methyl dichloroacetate were~added. The mixture was stirred for 24 hours and then evaporated under vacuum. The residue was purified by silica gel chromatography, using as solvent a mixture of: methylene chloride/methyl alcohol/H20, 80:10:1. The fractions containing the /3-2-O-ethylglycoside of N-dichloroacetylneuraminic ethyl ester were gathered and evaporated under vacuum. The residue was crystallized from a mixture of 50 ml of methanol and 200 ml of ethyl ether. Yield: 85%.
Rf =0.49, chloroform/meth.anol, 80:20.
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 ~i-2-O-ethylglycoside of the dimethylamide of N-palmitoylneuraminic acid of the formula:
Trademark WO 94/03469 ~ ~ ~~ ~ ~ ~'"~ ~ PCT/US93/07307 HO
0 HJ ~ y0/'~ 0 /N
H / \G"~~
identified as ND37.
Examble 53 Antineuronotoxic effect of 1VD37 in vitro on cerebellar crranule cells : protective effect on exocrenous glutamate induced neurotoxic;itv MATERIALS AND METHODS
Cell cultures Primary cell cultures of cerebellar granule cells were prepared from 6 day old Sprague-Dawley rats.
Neurons were grown in 35 mm plates for 11-13 days and maintained in a humid environment (95% air and 5o CO2).
Cultures (3x106 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 1x10-2 M in dimethylsul:Eoxide (1% DMSO) and then diluted at various concentrations in Locke's solution (154 mM NaCl/5.6 mM KC1/ 3.6 mM NaHC03/2.3 mM CaCl2/1 mM
MgCl2/5.6 mM glucose/5 mM HEl?ES, pH 7.4). The following concentrations were tested: 5x10-6 M, 1x10-5 M, and 2x10-M.
Description of the exogenous ctlutamate neurotoxicitv model The cell culture medium was aspirated from the plates (and correctly maintained) . Plates were washed (3 x 2 ml) with Locke's solution, and solutions (1.5 ml) WO 214 i 6 l ~ PCT/US93/073Q7 containing the compound to be tested (concentrations from 5x10-6 M to 2x10-5 M) were added and incubated for 2 hours in an incubator at :37 °C (5% C02).
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 (controls) were added. Incubation with glutamate was performed for 15 minutes ai. room temperature (27 °C) .
Glutamate was removed, th~~ plates were washed with Locke's solution (2 x 2 ml), and then incubated in presence of the starting medium (correctly maintained) for 24 hours at 37 °C in an incubator (5% C02). At the end of the incubation, cellular viability was assayed via quantification by the MTT colorimetric test (Mosmann T.: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55: 63, 1983 and modified according to Skaper S.D. et al.: Death of cultured hippocampal pyramidal neurons induced by pathological activation of N-methyl-D-aspartate receptors is reduced by monosialogangliosides. J. Pharm. Exp. Ter. 259,1 452-457, 1991) . Data are e:Kpressed as % survival. The significance was calculated according to the Dunnett test.
Results The obtained results (Table 1) show that:
- ND37 has a marked antineuronotoxic activity:
the presence of the free compound in the incubation medium during the exposure t:o the glutamate toxin is not necessary. The neuroprotect:ive effect of ND37 is very high: at a concentration of 1x10-5 M there is a protection of about 63%, and the highest protection (about 83%) is reached at ~~ concentration of 2x10-5 M.

WO 94/03469 21416 "~ 9 PC'f/US93/07307 Table 1. Antineuronotoxic effect of ND37 in cerebellar granule cells: protective effect glutamateinduced on exogenous neurotoxicity Groups MTT % survival values 1) control 0.195 f 0.016100 2) glutamate 0.075 0.009 38 ~ 5 3) glutamate + ND37 (5x100.110 t 0.01537 ~ 5 6 M) (1x10 5 M) 0.123 0.006 * 63 3 (2x10-5 M) 0.326 0.016 * 83 6 Significance (Dunnett's test) * p < 0.01 (vs group 2) 5 Example 54 Antineuronotoxic effect of ND37 in vitro in cerebellar ctranule cells : protective e:Efect on exocrenous glutamate-induced neurotoxicity durincr cotreatment of cells with the active compound Cell cultures Primary cerebellar granule cells were prepared from 8 day old rats (Zivic Mi:Ller, Pittsburgh, PA, USA).
Neurons were cultivated in :35 mm plates for 7-8 days and 15 maintained in a humid environment (95% air and 5% C02).
Cultures (3x106 cells/plate) are mainly formed by granule cells (95%) with a small amount (5%) of glial cells (Gallo V. et al.: Se:Lective release of glutamate from cerebellar granule cells differentiating in 20 culture. Proc. Natl. Acad. Sci. USA 79, 7919-23, 1982).
Glial proliferation was prEwented by arabinofuranoside cytosine.
Derivative ND37 was solubilized at a concentration of 1x10-2 M in dimethylsu7.foxide (1% DMSO), and then diluted at various concentrations in Locke's solution (154 mM NaCl/5.6 mM KC1/ 3..6 mM NaHC03/2.3 mM CaCl2/1 mM
MgCl2/5.6 mM glucose/5 mM HEPES, pH 7.4). The following concentrations were tested: 5x10-6 M, 1x10-5 M, and 2x10 - 5 5 M.
Description of the exocte:nous glutamate neurotoxicitv model 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 (1.5 ml) containing the compound to be tested (concentrations from 5x10-6 M to 2x10-5 M) were added and incubated for 2 hours in an incubator at 37 °C (5% C02).
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 4x10-5 M) in 1.5 ml of Locke's solution (-Mg++) were added. The incubation was conducted for l5~minwtes (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% C02). At the end of the incubation, 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 9:19-427, 1990). 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 Trademark WO 94/03469 2 ~ 6 '~ ~ PCT/US93/07307 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.
After glutamate treatment, some neurons can degenerate and detach from the plates. The loss of cells was estimated by comparing the number of intact or degenerated neurons in a well defined field, which was photographed before and after 24 hours after application of glutamate. The percent of surviving neurons in 4 representative fields (magnification 40x) of each monolayer was determined by a researcher unaware of the experimental conditions, evaluating the FDA/PI color, and calculating it as follows:
FDA positive cel:Ls ___________________________.______________ x 100 FDA positive + PI positive ~~ loosened cells Data are expressed as % of surviving cells.
Significance was calculated using the Dunnett's test.
Results The data in Table 2 show that the neuroprotective effect of ND37 on glutamate 'toxicity is immediate, i.e. , 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.

WO 94/03469 ~ PCT/U593/0730' 1 '~ 9 58 Table Antineuronotoxic effect of ND37 during 2: cotreatment of exogenous glutamate on cerebellar granule cells (pro-tective effect) Groups ~ surviving cells N = 4 (average + s.e.) FDA/PI test 1) control 89 t 7.0 2) glutamate 22 _+ 1.5 3) glutamate + ND37 (1x10 S M) 34 _+ 2.0 (2x10 5 M) 70 + 6.0 (4x10-5 M) 88 ~ 4.5 Significance (Dunnett's test) * p < 0.01 (vs group 2) Example 55 In vivo effect of ND37 on cerebral damage induced by intracerebroventricular (icv) infection of N-methyl-D-aspartate in neonatal rats MATERIALS AND METHODS
Description of the model Experiments were performed on 7 day old neonatal rats weighing ca. 13 grams. At the 7th day, animals, after ether anesthesia, were lesioned by icv injection of 25 nmoles/~.1 of N-methyl-D-aspartate (NMDA), Sigma, St. Louis, MO, USA, according to the method described by McDonald et al. (McDonald J.W. et al.: Neurotoxicity of N-methyl-D-aspartate is markedly enhanced in developing rat central nervous system. Brain Res. 459, 200-203, 1988). The excitotoxin was solubilized in saline, and the pH was brought to 7 . 4 by adding 1N NaOH . The NMDA
injection (25 nmoles/~,1) was performed slowly (2 minutes) at the level of the right lateral ventricle utilizing a Hamilton syringe. Saline was administered to control animals (1 ~.1 icv).
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.

WO 94/03469 ~ ~ ,~'~ (~ PCT/US93/07307 The treatment was conducted performing two administrations:
- 1 hour before NMD;A injection;
- immediately after NMDA injection.
Experimental crroups 1. (n = 8) saline (1 ~l) + saline (1 ~,1) 2. (n = 14) NMDA (25 nmo:Les/~,l)+ saline (1 ~.1) 3. (n = 15) NMDA (25 nmoles/~,1)+ ND37 (1 mg/kg/ml) 4. (n = 15) NMDA (25 nmoles/~1)+ ND37 (3 mg/kg/ml) 5. (n = 15) NMDA (25 nmoles/~,1)+ ND37 (5 mg/kg/ml) The number of animals in each experimental utilized group (corresponding to the total number of animals, i.e., experiment 1 + exp eriment according to 2, Table 3) is indicated brackets.
in Parameters 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.
The statistical significance was evaluated using Dunnett's test.
Results The obtained results ('.Cable 3) show that:
- treatment with ND:37 is effective in reducing the brain damage induced by the excitotoxin (evaluated as the lowering of total brain weight;
- ND37 is significat:ively effective (p < 0.01) at a dose of 1 mg/kg sc.

WO 94/03469 PCT/US93/073(17 ~1 ~~6~ 9 Table 3 Antineurotoxic effect of ND37 in vivo_ evaluation of the protection of cerebral damage (total brain weight) induced by NMDA in neonatal rat Experiment n.l (vehicle = DMSO 1%) Groups Compounds Doses Total brain weight (mg/kg) (mg) 1. 4) control + saline 1017 (n =

2. 7) NMDA + saline 841 (n =

3. 8) NMDA + ND 37 1 960 (n =

4. 8) NMDA + ND 37 3 968 *
(n =

5. 8) NMDA + ND 37 5 959 (n =

Experiment n.2 Groups Compounds Doses Total brain weight (mg/kg) (mg) 1. 4) control + saline 1017 (n =

2. 7) IAA + saline 841 (n =

3. 7) NMDA + ND 37 1 1019 (n =

4. 7) NMDA + ND 37 3 977 (n =

5. 7) NMDA + ND 37 5 976 *
(n =

* p < 0.01 vs lesioned not treated (group 2) (In all experiments the standard deviation is less than 5%).
In 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.
Modulatorv in vitro effect of ND37 on release and/or uptake of excititatorv neurotransmitter in cerebellar 5 Qranule cells: determination of glutamate and aspartate content in t~otassium induced depolarization MATERIALS AND METHODS
Cell cultures Primary cell cultures of cerebellar granule cells 10 were prepared from 8 day old Sprague-Dawley rats.
Neurons were grown in 35 mm plates for 11-13 days and maintained in a humid environment (95% air and 5%
C02). Cultures (3x106 cells/plate) are mainly formed by granule cells (95~), with a small amount (5%) of. glial 15 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).

214:16'9 Glial proliferation was prevented by arabinofuranoside cytosine.
Derivative ND37 was sol.ubilized 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 KC1/ 3.6 mM
NaHC03/2.3 mM CaCl2/1 mM MgC7.2/5.6 mM glucose/5 mM HEPES, pH 7.4) .
Description of exogenous glutamate neurotoxicitv model The cell culture medium was aspirated from the plates. Plates were washed. (3 x 2 ml) with Locke's solution, and solutions (750 ~,1) 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% C02) both in the presence and absence of different depolarizing concentrations of KC1 (between 5 and 50 mM) . The incubation medium was gathered, filtered through a 0.2 ~ filter, and processed (220 ~,1) for the analysis of the amino acid content.
Parameters 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.
Results The obtained results show that 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). The compound, administered at closes up to 20 uM, did not modify the basal levels (controls) of extracellular glutamate and aspartate (Table 5), whereas it reduced by ca. 30% the extracellular glutamate induced by KC1 potassium already at a dose of 0.1 ~M (Table 4). ND 37 WO 94/03469 PCT/US93/073~

completely abolished the glutamate increase induced by KC1 in the culture medium at a dose of 20 ~,M (Table 5).
20 ~M ND73 completely abolished the extracellular increase of glutamate and aspartate induced by KC1 (15, 25, 30, and 50 mM). The extracellular values for glutamate and aspartate in cultures exposed to KC1 in the presence of ND37 were between 0.01 and 0.28% of the values obtained without the compound (Table 5).
The possible mechanism of action of ND37 may reside in the release and/or uptake of endogenous compounds.
Table 4: Dose dependent inhibitory action of ND37 (0.1 - 1 - 10 -uM) on extracellular glutamate increase induced by KC1 in primary cerebellar granule cells.
Extracellular $ KC1 Groups glutamate 50 mM
content (uM) 1) controls 0.04 t 00 2) KCl (50 17.40 t 0.19 100 mM) 3) KC1 (50 + ND37(0_1 12.60 0.57 72 mM) uM) 4) KC1 (50 + ND37(1 13.30 t 1.33 76 mM) uM) 5) KCl (50 + ND37(10 5.60 2.37 * 32 mM) uM) 6) KC1 (50 + ND37(20 0.11 ~ 0.09 * 0.01 mM) uM)-Triplicate (mean s.e.) experiments +

* p c 0.01 group vs. 2 (Dunnett's test) ~ ..,WO 94/03469 214 ;~ ~; ~ g 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 KC1 (15 - 50 mM).
Extracell_ % Extracell. % corresp.
corresp.

Groups glutamate KC1 aspartate KC1 content (uM) content (uM) 1)controls 0.354 0.020 2)controls +

ND37 (20 0.362 0.039 uM) 3)KC1 (30 23.115 :100 1.016 100 mM) KC1 (50 +
mM) ND37 (20 0.318 * 0.01 0.063 0.06 uM) 4)KC1 (50 10.863 :100 0.621 100 mM) KC1 (50 +
mM) ND37 (20 0.246 * 0.02 0.078 0.13 uM) 5)KC1 (25 5.195 :100 0.386 100 mM) KC1 (50 +
mM) ND37 (20 0.330 * 0.06 0.107 0.28 uM) 6)KC1 (15 2.459 100 0.484 100 mM) KC1 (50 +
mM) ND37 (20 0.899 * 0.34 0.046 0.09 uM) Triplicate experiments * p < 0.01 (Dunnet's test)corresponding treated vs group only with KC1 (in all experiments:>tandarddeviation than 5%) less Examp7Le 56 Electrophysioloctical characterization of neurons treated with ND37: absence of an effect on Qlutamate-stimulated cationic channels.
Materials and Methods Primary cell cultures of cerebellar granule cells were prepared from 8 day old rats (Zivic Miller, Pittsburgh, PA, USA).
Neurons were grown on 35 mm plates for 7-8 days and maintained in a humid environment (95% air and 5% C02).
Cultures (2.5x106 cells/plate) were mainly formed by granule cel is (95%) , Grith 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).

WO 94/03469 PCT/US93/073~' ~14~~~ 9 Glial proliferation was prevented by arabinofuranoside cytosine.
Primary cell cultures of cortical neurons were prepared from one day old neonatal mice (Zivic Miller, Pittsburgh, PA, USA) (Bertolino and Vicini: Mol.
Pharmacol. 34, 98-103, 1988).
Cultures were grown on 35 mm plates, to which was added 10 ~g/ml of poly-L-lysine, at a density of 5x105 cortical neurons/plate. Cultures were prepared on basal Eagle medium (Gibco) containing loo fetal calf serum (Gibco), 25 mM KC1, 2 mM glutamine, and 100 ~g/ml gentamycine . Glial proliferation was prevented by adding 1 ~M arabinofuranoside cytosine 24 hours after seeding.
Cortical neurons were cultivated for 3 weeks.
Derivative ND37 was solubilized at a concentration of 1x10-2 M in dimethylsulfoxide (1% DMSO), and then diluted at various concentrations in Locke's solution (-Mg++) (154 mM NaCl / 5.6 mM KC1 / 3.6 mM NaHC03 / 2.3 mM CaCl2 / 5 . 6 mM glucose / 5 mM HEPES , pH 7 . 4 ) . ND3 7 was analyzed at concentrations of 20 and 30 ACM.
Description of electrophysiological measurements of glutamate-related cationic channel activity in cerebellar granule neurons and cortical neurons in culture Experiments were performed at room temperature utilizing the following medium 145 mM NaCl, 1 mM CaCl2, 5 mM glucose, and 5 mM HEPES/NaOH at pH 7.4, and patch pipettes, close to the preparation, containing 145 mM
CsCl2, 1 mM CaCl2, 11 mM ethyleneglycol (~i-aminoethyl ether) bis N,N'-tetraacetic acid, 10 mM HEPES/Cs(OH)2 at pH 7.2. Plates were continually perfused at a flow rate of 1 ml/min.
a) "Sealed" registrations on whole cells (Hamill O.P. et al., "Improved patch-clamp techniques for high-resolution current recording from cells and _yy0 94/03469 2141 ~6 /~ ~ PCT/US93/07307 E~ 5 cell-free membranes patches", Pfluegers Arch. 391, 85-100,1981) were performed on cerebellar granule cells or cortical neurons from neonatal rats and grown in cultures for 1 or 2 weeks, respectively.
Glutamate was released. under pressure (2 - 6 psi) from glass micropipettes (with tips having a greater delivery capacity than that, 4 - 6 ~M, of patch pipettes) on the cell bodies of voltage clamped neurons (maintenance voltage - 40 mV).
b) The activation by glutamate of single channels was externally recorded on membrane portions prepared from cerebellar granule cells pretreated with ND37 for 30 minutes at 37 °C. Micropipettes, filled (1 ~,M) with glutamate, were positioned close to the membrane portions and were also utilized in order to drip the neurotransmitter. Single channel currents were registered according to the method described by Bertolini and Vicini ("Voltage-dependent block by strychnine of N-methyl-D-aspartic acid-activated cationic channels in rat cortical neurons in culture", Mol. Pharmacol. 34, 98-103, 1988).
Registration on the whole cell 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 respon;ae 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 oontrols (mean 200 pA at voltage - 50 mV) in the three different conditions in 3 cerebellar <~nd 5 cortical preparations.

WO 94/03469 PCT/US93/07z~' Results In order to evaluate the activities of ND37 on glutamate response, 10 different experiments were performed. In cerebellar granule cell membrane preparations, high conductivity, glutamate-activated (50 pS) cationic channels are prevalent. Pretreatment of cells for 30 minutes with 20 ~.M ND37 does not influence the channel conductivity and the frequency of their opening (Table 6). According to the single channel registration, no kinetic and conductivity variations were observed after ND37 treatment. The inside currents activated by glutamate (50 ~,M) given contemporaneously with ND37 (30 ~.M) were not different from those activated only by glutamate.
Table 6: Effect of ND37 on glutamate controlled cationic channels activity in cerebellar granule neurons and cortical 1 5 neurons in culture.
Single channel current registration Scerebellar granule neurons) Group n = 10 Channel conductivity Opening frequence (pS) (opening/sec) 1) Glutamate (1 mM) SO 4.5 + 2.9 2) Glutamate + ND37 (20 uM) 50 4.1 + 2.1 Conclusions 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 ~WO 94103469 2 ~ 4 ~ s 7 ~ PCT/US93/07307 performed with the aforesaid compound ND37, and with the 2-ethylglycoside of N-:palmitoyl-neuraminic acid dimethylaminopropylamide, which will be subsequently referred to as ND35.
MATERIALS AND METHODS
Cell cultures C1300 mouse neuroblast:oma cells, Neuro-2a clone, (American Cell Type Culturfs 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 ~,1 of fresh medium plus compounds to be tested.
Tested compounds and their solubilization Compounds ND35 and ND37 were solubilized at a concentration of 1x10-2 M in dimethylsulfoxide (1% DMSO) .
For the different compounds, progressive dilutions in culture medium were performed (concentrations from 1x10-5 to 1x10-4 M).
Parameters NeuritoQenic 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 ne:urites was determined after counting of at least 100 cells (Facci L. et al.:
Promotion of neuritogenesis in mouse neuroblastoma cells by exogenous ganglioside GM7_. J. Neurochem. Raven Press, New York, 299-305,1984).

WO 94/03469 ' PCT/US93/073°' ~r Results The obtained results (Table 7) show that derivatives ND35 and ND37 both induce neuritogenesis in vitro. In particular, under the tested experimental conditions, it was shown that:
- the neuritogenic effect was already significant at a concentration of 5x10-5 M (p < 0.01), with the highest efficacy (about 480 of cells with neurites) at a concentration of 1x10-4 M.
Table 7: Neuritogenic effect of ND35 and ND37 in N2a neuroblastoma cells.
Compounds (concentrations) Cells with neurites (%) Control 2 _+ 3 ND37 (1x10 4) 48 _+ 9 (5x10 5) 27 _+ S
(2.5x10 5) 8 _+ 4 (1x10 5) 5 + 3 ND35 (1x10 4) 39 _+ 7 *
(5x10 5) 28 _+ 7 *
(2.5x10 5) 7 _+ 3 (1x10 5) 7 + 2 * p < 0.01 vs. control (Dunnett's test) -~..WO 94/03469 ~ ~ ~ ~ ~ ~ PCT/US93/07307 CONCLUSIONS
The foregoing results show a good pharmacological profile of the compounds according to the present invention. Their antineuronotoxic and modulatory effects on extracellular levels of e:r~cititatory neurotransmitter amino acids should be noted in particular.
Owing to their antineuronotoxic activity, 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., glutami.c 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. Although 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. Damage to the nervous system due to excititatory amino acids is present in ischaemia, h~~rpoxia, epilepsy, trauma, compressions, metabolic disfunctions, aging, and toxic-infective and chronic neurodegenerative diseases, like Alzeimer's and Huntingt:on's diseases.
Because of the modu7.atory 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 exci.titatory amino acids occurs through the activation of glutamate receptors, the use of these compounds does not. have the disadvantages of .. ,0 2141 a ~ 9 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 neu:rotoxicity: mechanism and prevention", Science 254, 1515-1518, 1991).
Finally, 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.
PHARMACEUTICAL APPLICATIONS OF THE COMPOUNDS
OF THE PRESENT INVENTION
Objects of the pre:>ent 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 be used for oral, rectal, parentera.l, 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. For parenteral use, it is possible to use formulations for int:ramuscular, 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. For local application, spray preparations, e.g., nasal sprays, can be employed, or ointments for topical use, or plasters for transdermal administration can be used..

,1 2141679 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 so7lid form preparations.
The dosage will vary according to the indication, the desired ei=fect, and the route of administration. For therapeutic administration, or for prophylaxis by the parenteral route, 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.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims (69)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A compound selected from the group consisting of a carboxylic amide of a compound of formula I:
wherein the C-1 carbon is amidated and wherein Ac represents an acyl residue of an aliphatic, araliphatic, aromatic, alicyclic or heterocyclic carboxylic acid, with the proviso that said aliphatic carboxylic acid is not acetic acid, a 2-hydrocarbyl-glycoside of said amide, a peracylated derivative of said amide wherein position 2 is in the .alpha. position, a basic addition salt of one of the foregoing compounds, and an acidic addition salt of one of the foregoing compounds having an acidic or basic function.
2. A carboxylic amide according to claim 1, wherein said acyl residue Ac has at least 4 and not more than 24 carbon atoms derived from a substituted or non-substituted acid, one to three substituents selected from the group consisting of: a halogen atom, a free, esterified, or etherified hydroxylic or mercapto group, a free or esterified carboxylic or sulfonic group, a free or esterified carboxylic or sulfonic group transformed into an amide, a free or amino group-substituted hydrocarbylic group, and a hydrocarbylic group interrupted by an -SO-, -SO2-, or phenylene group.
3. A carboxylic amide according to claim 2 wherein said halogen atom is a member selected from the group consisting of fluorine, chlorine and bromine.
4. A carboxylic amide according to claim 2, wherein said esterified hydroxylic or mercapto group is derived from an aliphatic or aromatic acid with not more than 8 carbon atoms.
5. A carboxylic amide according to claim 2, wherein said etherified hydroxy or mercapto group, or said esterified carboxylic or sulfonic group, is derived from an aliphatic alcohol with not more than 8 carbon atoms, or from an araliphatic alcohol with only one benzene ring and an alkylene of 1 or 2 carbon atoms.
6. A carboxylic amide according to claim 2, wherein said hydrocarbylic group which substitutes said amino group is an alkyl with at least 8 carbon atoms or an aralkyl with one benzene ring and an alkylene of 1 or 2 carbon atoms.
7. A carboxylic amide according to claim 2, wherein said acyl group Ac is saturated.
8. A carboxylic amide according to claim 1, wherein said acyl group Ac is unsaturated and contains only one double bond.
9. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from an acid selected from the group consisting of butyric, valeric, caproic, enantic, caprilic, pelargonic, capric, undecilic, di-tert-butyl-acetic, 2-propyl-valeric, lauric, tridecilic myristic, pentadecilic, palmitic, margaric, stearic, arachic, behenic and lignoceric acid.
10. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from an acid selected from the group consisting of levulinic succinic, valine, leucine, phenylalanine, tryptophan, .alpha.-aminobutyric, .beta.-aminobutyric, methionine, lysine, aspartic, glutamic, proline and hydroxyproline.
11. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from mono- or dichloro-butyric acid, trichloro-butyric acid or tribromo-butyric acid.
12. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from a natural or synthetic peptide having not more than 12 carbon atoms.
13. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from a peptide contained in the thymus gland.
14. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from phenylacetic cinnamic, phenylpropionic or atropic acid.
15. A carboxylic amide according to claim 1, wherein said acyl group Ac is derived from benzoic acid and its methylated homologues, salicylic acid, anthranilic acid, trimethoxybenzoic acid, phthalic acid, terephthalic acid, diphenyl-o,o'-dicarbonic acid, chloro-benzoic acid, vanillic acid, veriatric acid, or piperonilic acid.
16. A carboxylic amide according to claim 1, wherein said aryl group Ac is derived from a heterocyclic acid selected from the group consisting of nicotinic, isonicotinic, cinconninic, lysergic, isolysergic, dihydrolysergic, 2-bromo-lysergic, 2-bromo-dihydrolysergic, 1-methyl-lysergic, 1-methyl-dihydro-lysergic, 1-methyl-2-bromo lysergic, and teophyllinacetic acide.
17. A carboxylic amide according to claim 1, having an amido group which is a non-substituted amide, -CONH2, or is derived from a primary or secondary aliphatic, aromatic, araliphatic, alicyclic or heterocyclic amine having not more than 24 carbon atoms.
18. A carboxylic amide according to claim 1 having a hydrocarbylic residue which is substituted with one to three groups selected from the group consisting of a free, esterified or etherified hydroxylic or mercapto group, a halogen atom, a free, esterified, or amide-modified carboxylic or sulfonic group, a free amino group, and a hydrocarbyl-substituted amino group wherein said hydrocarbyl group is blocked with an -SO- or -SO2- group.
19. A carboxylic amide according to claim 18, wherein said esterified hydroxy or mercapto group is derived from an aliphatic or aromatic acid having not more than 8 carbon atoms.
20. A carboxylic amide according to claim 18, wherein said esterified hydroxylic or mercapto group, or said esterified carboxylic or sulfonic group, is derived from an aliphatic alcohol having not more than 8 carbon atoms, or from an araliphatic alcohol having only one benzene ring and an alkylene of 1 or 2 carbon atoms.
21. A carboxylic amide according to claim 18, wherein said hydrocarbylic residue substituting said amino group is an alkyl having not more than 8 carbon atoms, or an aralkyl with only one benzene ring and an alkylene of 1 or 2 carbon atoms.
22. A carboxylic amide according to claim 17, wherein said amido group is derived from an alkyl- or dialkylamine having between 1 and 12 carbon atoms.
23. A carboxylic amide according to claim 17, wherein said amido group is derived from an alkylenamine having between 3 and 6 carbon atoms constituting a ring.
24. A carboxylic amide according to claim 17, wherein the amidic group derives from methylamine ethylamine, propylamine, hexylamine, diethylamine, dimethylamine, diisopropylamine, dihexylamine pyrrolidine, pyperidine, or azepine.
25. A carboxylic amide according to claim 17, wherein the amido group derives from an aliphatic diamine.
26. A carboxylic amide according to claim 25, wherein said diamine is selected from the group consisting of ethylenediamine, trimethylenediamine, tetramethylenediamine, pentamethylenediamine, hexamethylenediamine, piperazine, and its N-alkyl or C-alkyl derivatives having a C1-4 alkyl.
27. A carboxylic amide according to claim 17, wherein the amido group is derived from aminoethanol, aminopropanol, mercaptoethylamine, morpholine, tiomorpholine, and a C1-4 alkylated derivative thereof.
28. A carboxylic amide according to claim 17, wherein the amido group is derived from an amino acid selected from the group consisting of valine, leucine, phenylalanine, tryptophan, .alpha.-aminobutyric acid, .beta.-aminobutyric acid, methionine, lysine, aspartic acid glutamic acid, proline, and hydroxyproline.
29. A carboxylic amide according to claim 17, wherein the amido group is derived from a natural or synthetic peptide having not more than 12 carbon atoms.
30. A carboxylic amide according to claim 17, wherein the amido group is derived from a peptide contained in the thymus gland.
31. A carboxylic amide according to claim 17, wherein the amido group is derived from an amine selected from the group consisting of phosphatidyl-ethanolamine, phosphatidylserine, sphingosine, dihydrosphingosine, psychosine, dihydropsychosine, phosphorylcholine-sphingosine, phosphorylcholine-dihydrosphingosine, and phytosphgosine.
32. A carboxylic amide according to claim 17, wherein the amido group is derived from an aromatic amine having only one non-substituted aromatic ring, or an aromatic amine substituted with one to three functional groups selected from the group consisting of a halogen, a hydroxylic or methoxylic group, a carboxylic or sulfonic group, and a C1-4 lower aliphatic hydrocarbylic group.
33. A carboxylic amide according to claim 17, wherein the amido group is derived from a heterocyclic amine selected from the group consisting of a pyrimidine base, a purine, ephedrine, tyramine, and adrenaline.
34. A carboxylic amide according to claim 1, wherein the 2-hydrocarbyl-glycoside is derived from an aliphatic alcohol having not more than 12 carbon atoms.
35. A carboxylic amide according to claim 1, wherein the 2-hydrocarbyl-glycoside is derived from an araliphatic alcohol having only one non-substituted benzene ring, or a benzene ring substituted wth 1 - 3 lower C1-4 alkyl groups, and having not more than 4 carbon atoms in the aliphatic chain.
36. A carboxylic amide according to claim 1, wherein the 2-hydrocarbyl-glycoside is derived from an alicyclic alcohol or an aliphatic-alicyclic alcohol having only one cycloaliphatic ring and not more than 14 carbon atoms.
37. A carboxylic amide according to claim 1, wherein the 2-hydrocarbyl-glycoside is derived from a heterocyclic alcohol having not more than 12 carbon atoms, and only one heterocyclic ring containing 1 or 2 heteroatoms selected from the group consisting of -NH-, -O- and -S-.
38. A carboxylic amide according to claim 1, wherein the 2-hydrocarbyl-glycoside is derived from a corticosteroid alcohol.
39. A carboxylic amide according to claim 1, wherein in the peracylated derivatives, the acyl groups are derived from aliphatic acids having not more than 10 carbon atoms.
40. A carboxylic amide according to claim 1, wherein in the peracylated derivatives, the aryl groups are derived from aromatic acides having only one benzene ring.
41. The .beta.-2-O-ethylglycoside of N-palmitoyl-neuraminic acid.
42. The .beta.-2-O-ethylglycoside dimethylamide of N-palmitoyl-neuraminic acid.
43. The .beta.-2-O-ethylglycoside arginylamide of N-acetyl-neuraminic acid.
44. The .alpha.-2-O-ethylglycoside arginylamide of N-acetyl-neuraminic acid.
45. The .beta.-2-O-ethylglycoside L-alanyl-D-isoglutaminylamide of N-palmitoylneuraminic acid.
46. The .beta.-2-O-ethylglycoside L-alanyl-D-isoglutaminylamide of N-acetylneuraminic acid.
47. The .alpha.-2-O-ethylglycoside L-alanyl-D-isoglutaminylamide of N-acetylneuraminic acid.
48. The .alpha.-2-O-ethylglycoside dimethylamino-propylamide of N-acetylneuraminic acid.
49. The .alpha.-2-O-ethylglycoside butylamide of N-acetylneuraminic acid.
50. The .beta.-2-O-ethylglycoside dimethylamide of N-acetylneuraminic acid.
51. The .beta.-2-O-ethylglycoside dimethylamino-propylamide of N-acetylneuraminic acid.
52. The .beta.-2-O-ethylglycoside benzylamide of N-acetylneuraminic acid.
53. The .beta.-2-O-ethylglycoside butylamide of N-acetylneuraminic acid.
54. The butylamide of N-acetylneuraminic acid.
55. A compound selected from the group consisting of a peracylated derivative of a carboxylic ester of the compound of the following formula:
wherein the C-1 carbon is amidated and wherein Ac represents any acyl residue of an aliphatic, araliphatic, aromatic, alicyclic, or heterocyclic carboxylic acid, and a basic or acidic addition salt thereof having an acidic or basic function.
56. A process for preparing a carboxylic amide of N-acylneuraminic acid of claim 1, comprising in a stepwise manner, converting the carboxylic acid of the N-acyl neuraminic acid into the C-1 amide function, optionally introducing the 2-glycosidic group to the N-acyl neuraminic acid, and optionally introducing the ester function and acyl, groups into the hydroxy groups.
57. The process according to claim 56, further comprising forming a salt of said carboxylic amide.
58. The process according to claim 56, wherein the carboxylic group of said N-acylneuraminic acid or of a 2-hydrocarbyl glycosidic derivative thereof is transformed into an amidic group.
59. The process according to claim 56, wherein the carboxylic group of said neuraminic acid is converted into the amide, and the free amino group is acylated with the desired acid.
60. The process according to claim 59, wherein the 2-hydroxy group in the hydrocarbyl derivative of said neuraminic acid is converted into the desired amide.
61. The process according to claim 59, wherein the free hydroxy groups are peracylated.
62. The process according to claim 59, wherein the peracylation is performed in any step of the process.
63. A process for preparing a compound according to claim 55, comprising introducing, in a stepwise manner, an ester function and acyl groups into hydroxy groups and of said compound.
64. The process according to claim 63, further comprising introducing a 2-glycosidic group into said N-acyl-neuraminic acid.
65. The process according to claim 64, further comprising forming salts of said compound.
66. Use of the compound according to claim 1 for therapies of diseases and disorders of the central nervous system.
67. The use according to claim 66, wherein said diseases and disorders of the central nervous system are selected from the group consisting of ischemia, hypoxia, epilepsy, trauma anal compressions, metabolic dysfunctions, aging, and toxic-infective and neurodegenerative diseases.
68. A pharmaceutical composition comprising a compound according to claim 1 as active ingredient, and a pharmaceutically acceptable carrier.
69. The process according to claim 58, further comprising converting the compound obtained therein into a peracylated derivative at the hydroxy groups therein.
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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 (en) * 1994-03-21 1997-06-04 Lifegroup Spa GLUCOSIDIC DERIVATIVES OF N-ACYL ALCHYLAMINES CAPABLE OF EXERCISING EFFECT OR NEUROPROTECTIVE USABLE IN ACUTE AND CHRONIC PATHOLOGIES OF THE CENTRAL NERVOUS SYSTEM RELATED TO EXCITOTOXICITY
US5849709A (en) * 1995-05-10 1998-12-15 Glycomed Incorporated Saccharopeptides and derivatives thereof
EP0837070B1 (en) * 1995-06-23 2001-10-31 Mitsubishi Chemical Corporation Sialic acid derivatives
US5824662A (en) * 1996-09-27 1998-10-20 Guilford Pharmaceuticals Inc. Treatment of global and focal ischemia using naaladase inhibitors
IL128718A0 (en) 1996-09-27 2000-01-31 Guildford Pharmaceuticals Inc Naaldase compositions
CA2328085C (en) 1998-04-10 2006-08-22 Mitsubishi Chemical Corporation Solid dispersion containing sialic acid derivative
CN1788722A (en) * 2001-05-23 2006-06-21 钱吉子 Composition for preventing or treating dementia
JP4005115B1 (en) * 2007-02-08 2007-11-07 日本臓器製薬株式会社 Pain disease treatment
JP5327839B2 (en) * 2008-06-09 2013-10-30 国立大学法人埼玉大学 Method for producing sialic acid derivative and its use as an influenza virus inhibitor
JP5298390B2 (en) * 2009-11-19 2013-09-25 学校法人福岡大学 Fluorosylated sialic acid derivative and analysis method thereof
TWI599354B (en) 2016-08-10 2017-09-21 國立臺灣師範大學 Use of tetrahydropyranol derivatives for manufacturing pharmaceutical composition of tau-associated diseases

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 (en) * 1984-05-01 1985-11-18 Kanto Ishi Pharma Co Ltd N-substituted neuraminic acid derivative and its preparation
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 (en) * 1989-11-06 1991-06-27 Mect Corp Sialic acid-bonded physiologically active peptide and production thereof

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WO1994003469A1 (en) 1994-02-17
ITPD920146A1 (en) 1994-02-03
ITPD920146A0 (en) 1992-08-03
CA2141679A1 (en) 1994-02-17
IT1260156B (en) 1996-03-28

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