CA2220771A1 - Antibiotics - Google Patents
Antibiotics Download PDFInfo
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- CA2220771A1 CA2220771A1 CA002220771A CA2220771A CA2220771A1 CA 2220771 A1 CA2220771 A1 CA 2220771A1 CA 002220771 A CA002220771 A CA 002220771A CA 2220771 A CA2220771 A CA 2220771A CA 2220771 A1 CA2220771 A1 CA 2220771A1
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- Canada
- Prior art keywords
- alkyl
- polyene macrolide
- salts
- antibiotics
- methyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The invention relates to N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group of general formula (1a), wherein M represents polyene macrolide antibiotics residues, R represents a variable part of sugar residue, and each of R1 and R2 represents a C1-4 alkyl group; their salts of general formula (2a), wherein R, R1, R2 and M are as herein defined and A represents an anion of organic or inorganic acid.
Compounds of formula (1b) and (2b) wherein the N-alkyl and alkyl ester groups are represented by methyl are of particular interest. The invention relates also to processes for preparation of the compounds, to compositions containing these compounds and to their use in therapy and the treatment of external and internal fungal infections in humans and animals.
Compounds of formula (1b) and (2b) wherein the N-alkyl and alkyl ester groups are represented by methyl are of particular interest. The invention relates also to processes for preparation of the compounds, to compositions containing these compounds and to their use in therapy and the treatment of external and internal fungal infections in humans and animals.
Description
1- ~137'.156\Pl,SPEC'~PC'rAMI
ANTIBIOTICS
The invention relates to N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of the polyene macr~lide group of general formula I (a), wherein M represents polyene macrolide antibiotics residues, R represents a variable part of sugar residue, and each of R1 and R2, which may be the same or different, represents a C 1-~ alkyl group; to 5 salts of compound l(a), represented by the general formula 2(a), wherein M, R, R1 and R2 are as defined above and A represents an anion of an inorganic or organic acid; to methods of preparation of compounds of general formula l(a) and 2(a) and their use in medicine.
\,~/ ~ ~
l(a) Compounds of particular interest include N-methyl-N-glycosyl derivatives of me~hyl 10 esters of antifungal antibiotics of polyene macrolide group of general formula 1 (b), wherein M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and their salts of general formula 2(b), wherein M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and A represents an anion of inorganic or organic acid, and to methods of their ~cp~Lion, and 15 use in medicine.
N-alkyl derivatives of antibiotics of polyene macrolide group wherein the amino group of the parent antibiotic is substituted by an alkyl group are known.
N-glycosyl derivatives of polyene macrolides wherein amino group of the parent àntibiotic is s~lbstihltçd with a residue of sugar are also known (J. Antibiotics 28, 244 20 (1975), L. Falkowski, J. Golik, P. Kolodziejczyk, J. Pawlak, J. Zielinski, T. 7:imin~ki, E.
Borowski; Acta Polon. Pharm. 37, 517 (1980), L. Falkowski, J. Pawlak, J. Golik, P.
Kolodziejczyk, B. Stef~n~L ~, E. Bylec, E. Borowski). Examples of sugars used in the pL~a.~tion of these derivatives include D-glucose, D-mannose, L-rhamnose, D-ribose and maltose. Upon reaction of polyene macrolides with the appl~.iate sugar a simultaneous 25 Amadori rearrangement occurs to give the corresponding N-glycosyl derivatives. The ~,~ 0 S~
r ~ l 3, 1517~PL.SP~Cl~CTA:.,tl compounds have the advantage that they exhibit a biological activity similar to those of the starting antibiotics and form water soluble salts. However, the high toxicity associated therewith represents a significant disadvantage.
From papers J. Antibiotics 28, 244 ( 1975), L. Falkowski, J. Golik, P. Kolodziejczyk, 5 J. Pawlak. J. Zielinski, T. 7imin~ki, E. Borowski; Acta Polon. Pharm. 37, 517 (1980), L. Falkowski, J. Pawlak, J. Golik, P. Kolodziejczyk, B. Stefanska, E. Bylec, E. Borowski) and US 4,093,796, US 5,314,999; US 4,0027741 and US 4,195,172, there are known N--glycosyl derivatives of polyene macrolides, in which the amino group of the parent antibiotic is substituted with a residue of sugar, such as D-glucose, D-mannose, L-10 rhamnose, D-ribose, maltose. The compounds are prepared by reaction of polyene macrolides with the listed sugars, and simlllt~neous Amadori rearrangement. The compounds exhibit the biological activity similar to those of the starting antibiotics and form water soluble salts. However, they exhibit high toxicity.
Complexes of the N-glycosyl derivatives of the polyene macrolide antibiotics with I S N-methylglucosamine (US 5,314~999), meglumine (US 4,002,741 ) and N-methylgln ~ ine (US 4,195,172) have been prepared. These complexes have also been found to have anti-fungal activity. However the meglumine and N-methylglll~mine complexes have the disadvantage of being relatively toxic. Upon formation of the complexes the nitrogen of the N-methylglucosamine, the meglumine or the N-methylgluc~mine is protonated by the20 carboxylic acid group of the polyene macrolide antibiotic.
Trimethylammonium derivatives of polyene macrolides methyl esters wherein the amino group of the parent antibiotic is fully metnylated to give a quaternary ammonium salt are also known (J. Antibiotics 32, 1080 (1979), L. FaLkowski, B. Stefanska, J. Zielinski, E.
Bylec, J. Golik, P. Kolodziejczyk, E. Borowski). The compounds are prepared by 25 exhaustive methylation of the parent antibiotic with dimethyl sulphate. Advantageous properties of the derivatives include their solubility in water and an antifungal activity similar to that of the starting antibiotics. Unfortunately they are very toxic and unstable.
N,N,N-trimethyl derivatives of polyene amphoteric antibiotics have been prepared(US 4,144,328) and have been found to have antifungal properties. However, associated 30 with these compounds is an undesirable level of toxicity.
Another type of derivatives are trimethylammonium derivatives of polyene ~ S~
1~ ~137'~56\PLSPEC\PCTAUI
macrolides methyl esters, which are known from a paper - J. Antibiotics 32, 1080 (1979), L. Falkowski, B. S~ef~n~k~ J. Zieliriski, E. Bylec, J. Golik, P. Kolodziejczyk, E. Borowski and in these compounds the amino group of the antibiot~c methyl esters is fully methylated to give quaternary ammonium salt. The compounds are prepared by exhaustive 5 methylation of the parent antibiotic with dimethyl sulphate. The derivatives are soluble in water and are characterized by antifungal activity similar to the activity of the starting antibiotics, but they are very toxic and unstable.
Other types of N-alkyl derivatives include the N-succinimidyl derivatives formed by Michael addition reaction of the antibiotics and N-substituted maleimides, such as:
10 N-ethylmaleimide, N,N'-hexamethylene~lim~leimide, N-(3-dimethylaminopropyl)-maleimide; the compounds are known from a paper - J. Antibiotics, 44, 979 (1991), A. Czerwinski, W.A. Konig, T. Zieniawa, P. Sowinski, V. Sinnwell, S. Milewski, E. Borowski. Such compounds are less toxic than the parent antibiotics, but their antifungal activity is ~limini~hed.
Finally, the last known group of N-alkyl derivatives of polyene macrolides are N-enamine and amidine derivatives, formed by reaction of the antibiotics with acetylacetone, ethyl acetyl~et~t~?, dimethylacetal or dimethylforrnamide; the compounds are presented in a paper - Acta Polonica Phann. 45, 71 (1988), B. Stefanska, J. Zieliriski, E. Borowski, L. Falkowski. The derivatives exhibit antifungal activity similar to those of 20 the parent antibiotics and improved solubility in organic solvents, however, they are still significantly toxic and very unstable.
The present inventors have now prepared mixed N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics. Methods of l~c~ ion of these mixed compounds have also been established. These novel compounds have been found to have 25 high anti-fungal activity, similar to those of the parent antibiotics; form water soluble salts with acids; and are ~ignifi-~ntly less toxic. These properties are unexpected since all of the N-alkyl derivatives of polyene macrolides of the prior art exhibit a high toxicity, which is a considerable disadvantage. The co..l~u,.ds comp r i~ed by the invention are devoid of this disadvantage.
Until now, N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolideantibiotics and methods of their ~,c~Lion were unknown. Surprisingly, such compounds "~'v''~' ~
r ~ C6~rLsPE~cTAM I
preserve high antifungal activity, similar to those of the parent antibiotics, they form water soluble salts with acids, and are dramatically less toxic. These compounds do not exhibit the same toxicity as the N-alkyl derivatives of the prior art. A high toxicity constitutes the basic dra~back of all known before N-alkyl derivatives of polyene macrolides, and the compounds comprised by the invention are devoid of this disadvantage.
A first aspect of the invention provides N-alkyl-N-glycosyl derivatives of alkyl esters of antibiotics of polyene macrolide group of general formula l(a), wherein M represents residue of an antibiotic of polyene macrolide group, R represents a part of sugar residue formed by reaction of the antibiotic with a mono or oligosaccharide, and each of Rl and R2, which may be the same or different, represents C 1-4 alkyl group.
M--~oOR2 \C/~~
~\ J
HO R
Formula l(a) Preferably the residue of the antibiotic of polyene macrolide group M is selected from amphotericin B, candidin, candidoin, c~n~ linin, mycoheptin, nystatin, polyfungin, aureofacin, vacidin, trichomycin or candicidin.
It is preferred that the mono or oligos~c~hs~ri<lç from which the sugar residue R is derived is selected from D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or m~ltosç Upon reaction of polyene macrolides with the ~pL~ L~ sugar a simlllt~n.-ous Amadori rearrangement occurs to give the N-glycosyl precursors to the compounds of the mvenhon.
A preferred embodiment of the invention comprises N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula I (b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligos~rh~ride, preferably with D-glu~ose7 or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by ~iml~lt~n.oous Amadori rearrangement.
1 '137~56\PLSPE,CI'CTA~II
~3C--N--CH2 o \C~ ) HO R
Forrnula l(b) ~ he invention in its preferred forrn relates to the derivatives wherein the antibiotic of polyene macrolide group is arnphotericin B, or candidin, or candidoin, or candidinin, or mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or 5 candicidin.
A second aspect of the invention provides salts of N-aL~cyl-N-glycosyl derivatives of antibiotics of polyene macrolide group of general formula 2(a) wherein M, R, R1 and R2 are as defined above for the first aspect of the invention and A represents an anion of an organic or inorganic acid. It is preferred that the salt is a physiologically acceptable salt and 10 compounds wherein A is the anion of L-aspartic acid are especially preferred. Salts wherein Rl and R2 are methyl groups are especially preferred.
R1--N--CH2 o A~
H "C~ ) HO R
Formula 2(a) A ~ler~ ,d embodiment of the second aspect of the invention comprises salts of N-methyl-N-glycosyl d~_,;v~liv~;s of antibiotics of polyene macrolide group presented by 15 general formula 2(b) wherein M leplcsenl~ residue of an antibiotic of polyene macrolide group, wherein R le~l~st;ll~ a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by cimlllt~n~ous Amadori rearrangement, and A
r ~l37\ 50\PLSPEC PCTAMI
represents an anion of organic or inorganic acid --HO R
Formula 2(b) The invention in its preferred form relates to the salts wherein the antibiotic of polyene macrolide group is amphotericin B, or candidin, or candidoin, or candidinin, or S mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or candicidin, and also preferably A relates to the anion of L-aspartic acid.
A third aspect of the invention comprises a method of ~1lep~dLion of compounds of formula 1 (a) wherein M, R, Rl and R2 are as defined above for the first and second aspects of the invention and which comprises the steps of reacting a polyene macrolide antibiotic with a mono or oligosaccharide, the reaction being characterised by the occurrence of a simultaneous Amadori rearrangement, to give the N-glycosyl derivatives of the polyene macrolide antibiotics; isolating the product of the Amadori rearrangement; treating the product with an alkylating agent and purifying the crude product.
In one embodiment of the third aspect of the invention the product of the Amadori rearrangement is isolated in the form of a suspension by precipitation from the solution in which the rearrangement occurs. Organic solvents such as N,N-dimethylformamide are preferred to support the Amadori rearrangement. Organic solvents such as diethyl ether are suitable to effect formation of a ~ el3~ion by precipitation of the product of the Amadori rearrangement.
In a second embodiment of the third aspect of the invention alkylation of the product of the Amadori rearrangement is carried out at reduced temperature. T~;lll~eld~ S of between -5~C and +5~C are ~L~r~ ;L Alkylating agents such as diazo alkanes may be used. An ethereal solution of diazomethane is the plef~ d alkylating agent.
Crude N-alkyl-N-glycosyl products can be isolated upon removal of the solvent and . . . .
1' '.137"~6~1'LSPEC~PCI'AMI
' ;', precipitation from diethyl ether. The pure product may be isolated using known purification procedures.
- A preferred embodiment of the third aspect of the invention comprises a process for preparation of the N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula l(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivatives of antibiotics of polyene macrolide group are transformed into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivatives in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -S ~C to +5 ~C, stirred, and isolated by evaporation of solvents, and precipitation I S from the concentrated solution, preferably by an e~cess of diethyl ether, then, the crude product is purified according to known procedures.
A fourth aspect of the invention provides a method of pLc:~dLion of salts of N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics of general formula
ANTIBIOTICS
The invention relates to N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of the polyene macr~lide group of general formula I (a), wherein M represents polyene macrolide antibiotics residues, R represents a variable part of sugar residue, and each of R1 and R2, which may be the same or different, represents a C 1-~ alkyl group; to 5 salts of compound l(a), represented by the general formula 2(a), wherein M, R, R1 and R2 are as defined above and A represents an anion of an inorganic or organic acid; to methods of preparation of compounds of general formula l(a) and 2(a) and their use in medicine.
\,~/ ~ ~
l(a) Compounds of particular interest include N-methyl-N-glycosyl derivatives of me~hyl 10 esters of antifungal antibiotics of polyene macrolide group of general formula 1 (b), wherein M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and their salts of general formula 2(b), wherein M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and A represents an anion of inorganic or organic acid, and to methods of their ~cp~Lion, and 15 use in medicine.
N-alkyl derivatives of antibiotics of polyene macrolide group wherein the amino group of the parent antibiotic is substituted by an alkyl group are known.
N-glycosyl derivatives of polyene macrolides wherein amino group of the parent àntibiotic is s~lbstihltçd with a residue of sugar are also known (J. Antibiotics 28, 244 20 (1975), L. Falkowski, J. Golik, P. Kolodziejczyk, J. Pawlak, J. Zielinski, T. 7:imin~ki, E.
Borowski; Acta Polon. Pharm. 37, 517 (1980), L. Falkowski, J. Pawlak, J. Golik, P.
Kolodziejczyk, B. Stef~n~L ~, E. Bylec, E. Borowski). Examples of sugars used in the pL~a.~tion of these derivatives include D-glucose, D-mannose, L-rhamnose, D-ribose and maltose. Upon reaction of polyene macrolides with the appl~.iate sugar a simultaneous 25 Amadori rearrangement occurs to give the corresponding N-glycosyl derivatives. The ~,~ 0 S~
r ~ l 3, 1517~PL.SP~Cl~CTA:.,tl compounds have the advantage that they exhibit a biological activity similar to those of the starting antibiotics and form water soluble salts. However, the high toxicity associated therewith represents a significant disadvantage.
From papers J. Antibiotics 28, 244 ( 1975), L. Falkowski, J. Golik, P. Kolodziejczyk, 5 J. Pawlak. J. Zielinski, T. 7imin~ki, E. Borowski; Acta Polon. Pharm. 37, 517 (1980), L. Falkowski, J. Pawlak, J. Golik, P. Kolodziejczyk, B. Stefanska, E. Bylec, E. Borowski) and US 4,093,796, US 5,314,999; US 4,0027741 and US 4,195,172, there are known N--glycosyl derivatives of polyene macrolides, in which the amino group of the parent antibiotic is substituted with a residue of sugar, such as D-glucose, D-mannose, L-10 rhamnose, D-ribose, maltose. The compounds are prepared by reaction of polyene macrolides with the listed sugars, and simlllt~neous Amadori rearrangement. The compounds exhibit the biological activity similar to those of the starting antibiotics and form water soluble salts. However, they exhibit high toxicity.
Complexes of the N-glycosyl derivatives of the polyene macrolide antibiotics with I S N-methylglucosamine (US 5,314~999), meglumine (US 4,002,741 ) and N-methylgln ~ ine (US 4,195,172) have been prepared. These complexes have also been found to have anti-fungal activity. However the meglumine and N-methylglll~mine complexes have the disadvantage of being relatively toxic. Upon formation of the complexes the nitrogen of the N-methylglucosamine, the meglumine or the N-methylgluc~mine is protonated by the20 carboxylic acid group of the polyene macrolide antibiotic.
Trimethylammonium derivatives of polyene macrolides methyl esters wherein the amino group of the parent antibiotic is fully metnylated to give a quaternary ammonium salt are also known (J. Antibiotics 32, 1080 (1979), L. FaLkowski, B. Stefanska, J. Zielinski, E.
Bylec, J. Golik, P. Kolodziejczyk, E. Borowski). The compounds are prepared by 25 exhaustive methylation of the parent antibiotic with dimethyl sulphate. Advantageous properties of the derivatives include their solubility in water and an antifungal activity similar to that of the starting antibiotics. Unfortunately they are very toxic and unstable.
N,N,N-trimethyl derivatives of polyene amphoteric antibiotics have been prepared(US 4,144,328) and have been found to have antifungal properties. However, associated 30 with these compounds is an undesirable level of toxicity.
Another type of derivatives are trimethylammonium derivatives of polyene ~ S~
1~ ~137'~56\PLSPEC\PCTAUI
macrolides methyl esters, which are known from a paper - J. Antibiotics 32, 1080 (1979), L. Falkowski, B. S~ef~n~k~ J. Zieliriski, E. Bylec, J. Golik, P. Kolodziejczyk, E. Borowski and in these compounds the amino group of the antibiot~c methyl esters is fully methylated to give quaternary ammonium salt. The compounds are prepared by exhaustive 5 methylation of the parent antibiotic with dimethyl sulphate. The derivatives are soluble in water and are characterized by antifungal activity similar to the activity of the starting antibiotics, but they are very toxic and unstable.
Other types of N-alkyl derivatives include the N-succinimidyl derivatives formed by Michael addition reaction of the antibiotics and N-substituted maleimides, such as:
10 N-ethylmaleimide, N,N'-hexamethylene~lim~leimide, N-(3-dimethylaminopropyl)-maleimide; the compounds are known from a paper - J. Antibiotics, 44, 979 (1991), A. Czerwinski, W.A. Konig, T. Zieniawa, P. Sowinski, V. Sinnwell, S. Milewski, E. Borowski. Such compounds are less toxic than the parent antibiotics, but their antifungal activity is ~limini~hed.
Finally, the last known group of N-alkyl derivatives of polyene macrolides are N-enamine and amidine derivatives, formed by reaction of the antibiotics with acetylacetone, ethyl acetyl~et~t~?, dimethylacetal or dimethylforrnamide; the compounds are presented in a paper - Acta Polonica Phann. 45, 71 (1988), B. Stefanska, J. Zieliriski, E. Borowski, L. Falkowski. The derivatives exhibit antifungal activity similar to those of 20 the parent antibiotics and improved solubility in organic solvents, however, they are still significantly toxic and very unstable.
The present inventors have now prepared mixed N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics. Methods of l~c~ ion of these mixed compounds have also been established. These novel compounds have been found to have 25 high anti-fungal activity, similar to those of the parent antibiotics; form water soluble salts with acids; and are ~ignifi-~ntly less toxic. These properties are unexpected since all of the N-alkyl derivatives of polyene macrolides of the prior art exhibit a high toxicity, which is a considerable disadvantage. The co..l~u,.ds comp r i~ed by the invention are devoid of this disadvantage.
Until now, N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolideantibiotics and methods of their ~,c~Lion were unknown. Surprisingly, such compounds "~'v''~' ~
r ~ C6~rLsPE~cTAM I
preserve high antifungal activity, similar to those of the parent antibiotics, they form water soluble salts with acids, and are dramatically less toxic. These compounds do not exhibit the same toxicity as the N-alkyl derivatives of the prior art. A high toxicity constitutes the basic dra~back of all known before N-alkyl derivatives of polyene macrolides, and the compounds comprised by the invention are devoid of this disadvantage.
A first aspect of the invention provides N-alkyl-N-glycosyl derivatives of alkyl esters of antibiotics of polyene macrolide group of general formula l(a), wherein M represents residue of an antibiotic of polyene macrolide group, R represents a part of sugar residue formed by reaction of the antibiotic with a mono or oligosaccharide, and each of Rl and R2, which may be the same or different, represents C 1-4 alkyl group.
M--~oOR2 \C/~~
~\ J
HO R
Formula l(a) Preferably the residue of the antibiotic of polyene macrolide group M is selected from amphotericin B, candidin, candidoin, c~n~ linin, mycoheptin, nystatin, polyfungin, aureofacin, vacidin, trichomycin or candicidin.
It is preferred that the mono or oligos~c~hs~ri<lç from which the sugar residue R is derived is selected from D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or m~ltosç Upon reaction of polyene macrolides with the ~pL~ L~ sugar a simlllt~n.-ous Amadori rearrangement occurs to give the N-glycosyl precursors to the compounds of the mvenhon.
A preferred embodiment of the invention comprises N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula I (b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligos~rh~ride, preferably with D-glu~ose7 or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by ~iml~lt~n.oous Amadori rearrangement.
1 '137~56\PLSPE,CI'CTA~II
~3C--N--CH2 o \C~ ) HO R
Forrnula l(b) ~ he invention in its preferred forrn relates to the derivatives wherein the antibiotic of polyene macrolide group is arnphotericin B, or candidin, or candidoin, or candidinin, or mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or 5 candicidin.
A second aspect of the invention provides salts of N-aL~cyl-N-glycosyl derivatives of antibiotics of polyene macrolide group of general formula 2(a) wherein M, R, R1 and R2 are as defined above for the first aspect of the invention and A represents an anion of an organic or inorganic acid. It is preferred that the salt is a physiologically acceptable salt and 10 compounds wherein A is the anion of L-aspartic acid are especially preferred. Salts wherein Rl and R2 are methyl groups are especially preferred.
R1--N--CH2 o A~
H "C~ ) HO R
Formula 2(a) A ~ler~ ,d embodiment of the second aspect of the invention comprises salts of N-methyl-N-glycosyl d~_,;v~liv~;s of antibiotics of polyene macrolide group presented by 15 general formula 2(b) wherein M leplcsenl~ residue of an antibiotic of polyene macrolide group, wherein R le~l~st;ll~ a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by cimlllt~n~ous Amadori rearrangement, and A
r ~l37\ 50\PLSPEC PCTAMI
represents an anion of organic or inorganic acid --HO R
Formula 2(b) The invention in its preferred form relates to the salts wherein the antibiotic of polyene macrolide group is amphotericin B, or candidin, or candidoin, or candidinin, or S mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or candicidin, and also preferably A relates to the anion of L-aspartic acid.
A third aspect of the invention comprises a method of ~1lep~dLion of compounds of formula 1 (a) wherein M, R, Rl and R2 are as defined above for the first and second aspects of the invention and which comprises the steps of reacting a polyene macrolide antibiotic with a mono or oligosaccharide, the reaction being characterised by the occurrence of a simultaneous Amadori rearrangement, to give the N-glycosyl derivatives of the polyene macrolide antibiotics; isolating the product of the Amadori rearrangement; treating the product with an alkylating agent and purifying the crude product.
In one embodiment of the third aspect of the invention the product of the Amadori rearrangement is isolated in the form of a suspension by precipitation from the solution in which the rearrangement occurs. Organic solvents such as N,N-dimethylformamide are preferred to support the Amadori rearrangement. Organic solvents such as diethyl ether are suitable to effect formation of a ~ el3~ion by precipitation of the product of the Amadori rearrangement.
In a second embodiment of the third aspect of the invention alkylation of the product of the Amadori rearrangement is carried out at reduced temperature. T~;lll~eld~ S of between -5~C and +5~C are ~L~r~ ;L Alkylating agents such as diazo alkanes may be used. An ethereal solution of diazomethane is the plef~ d alkylating agent.
Crude N-alkyl-N-glycosyl products can be isolated upon removal of the solvent and . . . .
1' '.137"~6~1'LSPEC~PCI'AMI
' ;', precipitation from diethyl ether. The pure product may be isolated using known purification procedures.
- A preferred embodiment of the third aspect of the invention comprises a process for preparation of the N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula l(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivatives of antibiotics of polyene macrolide group are transformed into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivatives in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -S ~C to +5 ~C, stirred, and isolated by evaporation of solvents, and precipitation I S from the concentrated solution, preferably by an e~cess of diethyl ether, then, the crude product is purified according to known procedures.
A fourth aspect of the invention provides a method of pLc:~dLion of salts of N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics of general formula
2(a) wherein M, R, R1 and R2 are as defined above for the first, second and third aspects of the invention and which comprises the steps of suspending an N-alkyl-N-glycosyl derivative prepared according to the third aspect of the invention in sufficient water to effect formation of a homogeneous suspension, acidifying the resulting suspension and isolating the product. The ~lepa.dlion of salts of N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolide antibiotics is especially pl~:f~ ,d.
Organic or inorganic acids may be used to acidify the suspension. L-aspartic acid is preferred.
Isolation of the pure product may be effected by precipitation of the crude product with an organic solvent which is then washed with an additional a~lo~l;ate solvent and dried. It is preferred that the solvent used to precipitate the crude product is miscible with water; acetone is pler~ d. Typical solvents employed for washing the product include acetone and diethyl ether. It is preferred that the product is dried under reduced pressure.
-7~
~,. .-, .~. . -~ ' 137.'JS-jil'LSPEC~PCT ~MI
A preferred embodiment of the fourth aspect of the invention comprises a process for preparation of salts of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose,~ or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivatives of antibiotics of polyene macrolide group are transformed into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5 ~C to +5 ~C, stirred, and isolated by evaporation of solvents, and precipitation lrom the concentrated solution, preferably by an excess of diethyl ether, then the crude product is purified according to known procedures, subsequently, the obtained derivative, as a solid, is suspended in small amount of water, and stoichiometric amount of organi~ or inorganic acid is added, next the product is precipitated from the formed solution by an excess of organic solvent miscible with water, preferably acetone, the solid is then washed, preferably with acetone and subsequently preferably with diethyl ether, and dried, preferably under reduced p~ ule A fifth aspect of the present invention comprises N-alkyl-N-glycosyl derivatives of formula I (a) or salts thereof for use in therapy.
A sixth aspect of the invention provides a method for the tr~?~trnent of fungal infections in humans and animals which comprises the administration thereto of N-alkyl-N-glycosyl derivatives of formula l(a) or salts thereof as herein before ~lefinefl N-methyl-N-glycosyl derivatives of formula 1 (b) or the salts thereof are of particular interest.
A first preferred embodiment of the sixth aspect of the invention comprises a method for treatment of external and internal fungal infections in humans and ~nim~l~, wherein N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group ~l~,s~llLed by general formula 1 (b), wherein M le~ sl;:llL ~ residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-F \ 13 7' ~ 5 6\PI,S PEC PCTAM I
mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement are used to treat the infections.
A second preferred embodiment of the sixth aspect of the invention comprises a method for treatment of external and internal fungal infections in humans and ~nim~
wherein salts of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anion of organic or inorganic acid are used to treat the infections.
A seventh aspect of the invention provides N-alkyl-N-glycosyl derivatives of formula I (a) or salts thereof as herein before defined for use in the treatment of fungal infections.
N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular I S interest.
The infections for which treatment is provided may be internal or external. The mode of administration will depend upon the nature of the infection. Thus the compounds of the invention may be form~ t~l for intravenous, intra peritoneal, oral, topical, subcutaneous, rectal or vaginal ~-lmini~tration.
A eighth aspect of the invention provides compositions for use in the treatment of fungal infections comprising an N-alkyl-N-glycosyl derivatives of formula 1 (a) or a salt thereof and a physiologically acceptable carrier. The nature of the N-alkyl-N-glycosyl derivative and the carrier will depend upon the mode of ~mini~tration. The composition may be form~ tsrl from one or more compounds according to the invention, optionally in combination with other known ~ntifilng~l agents, according to requirements. Compositions cont:~ining N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular interest.
An ninth aspect of the invention provides a unit dosage form comprising one or more N-alkyl-N-glycosyl derivative of formula l(a) or salts thereof and a physiologically acceptable carrier form~ te~1 for ph~rm~ceutical or V~ y use. The unit dosage form may be formlll~t~l from one or more compounds according to the invention optionally in g :.;~,.
, 56~PLSPE~PCTAMI
combination with other known antifungal agents. By analogy with the previous aspects of the invention unit dosage forms cont~ining N-methyl-N-glycosyl derivatives of formula I (b) are of particular interest.
As mentioned above the nature of the unit dosage form will depend upon the mode 5 of administration. Typically tablets and capsules are suitable for oral administration; crearns and patches are suitable for topical administration with pessaries being suitable for rectal and vaginal administration.
A tenth aspect of the invention provides the use of N-alkyl-N-glycosyl derivative of formula I (a) or salts thereof for the plepdldlion of a medicament for use in the treatment 10 of fungal infections. Compounds wherein the N-alkyl and alkyl ester substituents are both methyl groups are of particular interest.
Structural determinations carried out on the compounds of the invention using spectroscopic methods indicate that the integrity of the parent antibiotic is preserved during the reaction.
The invented process for ~repdldlion of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group gives the desired products without changes in structure of the parent antibiotic. Structure of the obtained compounds was proved using spectroscopic methods. The proof is illustrated by ~iet~-~nin~tion of the structure of N-methyl-N-D-fructosylarnphotericin B methyl ester, of formula 3.
/~CH3 ~ ~ H3 CN ~OH
J~CH3 OH OH OH OH o~CoocH3 OH
H3CJ~O ~OH
OH
= Forrnula 3 ~ d;~ "
1: '137'~56\PLSPECPCTAMI
Electronic spectrum of N-methyl-N-D-fructosylamphotericin B methyl ester is identical with that of the parent antibiotic, that means amphotericin B, and demonstrates that the invented method does not lead to degradation of the polyene chromophore, and a high value of e~tinction (El%lCm = 1300 at 382 nm) confirms ahigh purity ofthe obtained 5 product. Absorption infrared spectrum of the N-methyl-N-D-fructosylamphotericin B
methyl ester demonstrates the band related to stretching vibrations of the ester carbonyl group at 1730 cm~l, and lack of band of free carboxylic group, what means that the carboxylic group was completely transformed into methyl ester group. Complete information on structure of the N-methyl-N-D-fructosylamphotericin B methyl ester was 10 provided by nuclear magnetic resonance spectra (NMR) upon I H (DQF-COSY, ROESY), 13C (DEPT) and heterocorrelated spectra (Varian 300 MHZ spectrometer) allowed anassignment of the formula 3 for the compound.
The most significant 1 H and 13C information are listed in Tables 1 and 2, respectively. The NMR data for aglycone of amphotericin B are in full agreement with literature data - Magn. Reson. Chem.30,275, (1992), P. Sowiriski, J. Pawlak, E. Borowski, P. Gariboldi. 1 H chemical shifts (in DMSO/MeOD solvent system) of N-CH3 (o = 2.35 ppm) and H-1" (2.30 and 3.15 ppm) are characteristic for influence of an amino substituent. After acidification o changes to 2.92 ppm for N-CH3, and to 3.64 for H-1 " due to the protonation of the amino group (o for H-3' changes to 3.19 ppm). These data are 20 supported also by ROE effects between protons NCH3/H3', NCH3/H2', NCH3/Hl"b and I "a/H3'. Coupling constants and ROE indicate 4C 1 conformation of the mycosamine moiety, as it was found before for free arnphotericin B.
Table 1 ~l~S~ chemical shifts lH and ROE effects ofthe ~ crh~nde fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester.
1:'137~.56\PLSPECPCTAMI
Table 1 proton o[ppm] ROE for protons O[ppm] ROE for protons pyridine-d5 methanol-d49: I nMSO-d~: methanol-d., 4: 6 1' 4.77 2', 3', 5', 18b, COOMe 4.18 3', 5' 2' 4.41 1', 3', NMe, 17, COOMe, l"a? 4.04 3',NMe, COOMe
Organic or inorganic acids may be used to acidify the suspension. L-aspartic acid is preferred.
Isolation of the pure product may be effected by precipitation of the crude product with an organic solvent which is then washed with an additional a~lo~l;ate solvent and dried. It is preferred that the solvent used to precipitate the crude product is miscible with water; acetone is pler~ d. Typical solvents employed for washing the product include acetone and diethyl ether. It is preferred that the product is dried under reduced pressure.
-7~
~,. .-, .~. . -~ ' 137.'JS-jil'LSPEC~PCT ~MI
A preferred embodiment of the fourth aspect of the invention comprises a process for preparation of salts of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose,~ or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivatives of antibiotics of polyene macrolide group are transformed into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5 ~C to +5 ~C, stirred, and isolated by evaporation of solvents, and precipitation lrom the concentrated solution, preferably by an excess of diethyl ether, then the crude product is purified according to known procedures, subsequently, the obtained derivative, as a solid, is suspended in small amount of water, and stoichiometric amount of organi~ or inorganic acid is added, next the product is precipitated from the formed solution by an excess of organic solvent miscible with water, preferably acetone, the solid is then washed, preferably with acetone and subsequently preferably with diethyl ether, and dried, preferably under reduced p~ ule A fifth aspect of the present invention comprises N-alkyl-N-glycosyl derivatives of formula I (a) or salts thereof for use in therapy.
A sixth aspect of the invention provides a method for the tr~?~trnent of fungal infections in humans and animals which comprises the administration thereto of N-alkyl-N-glycosyl derivatives of formula l(a) or salts thereof as herein before ~lefinefl N-methyl-N-glycosyl derivatives of formula 1 (b) or the salts thereof are of particular interest.
A first preferred embodiment of the sixth aspect of the invention comprises a method for treatment of external and internal fungal infections in humans and ~nim~l~, wherein N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group ~l~,s~llLed by general formula 1 (b), wherein M le~ sl;:llL ~ residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-F \ 13 7' ~ 5 6\PI,S PEC PCTAM I
mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement are used to treat the infections.
A second preferred embodiment of the sixth aspect of the invention comprises a method for treatment of external and internal fungal infections in humans and ~nim~
wherein salts of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anion of organic or inorganic acid are used to treat the infections.
A seventh aspect of the invention provides N-alkyl-N-glycosyl derivatives of formula I (a) or salts thereof as herein before defined for use in the treatment of fungal infections.
N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular I S interest.
The infections for which treatment is provided may be internal or external. The mode of administration will depend upon the nature of the infection. Thus the compounds of the invention may be form~ t~l for intravenous, intra peritoneal, oral, topical, subcutaneous, rectal or vaginal ~-lmini~tration.
A eighth aspect of the invention provides compositions for use in the treatment of fungal infections comprising an N-alkyl-N-glycosyl derivatives of formula 1 (a) or a salt thereof and a physiologically acceptable carrier. The nature of the N-alkyl-N-glycosyl derivative and the carrier will depend upon the mode of ~mini~tration. The composition may be form~ tsrl from one or more compounds according to the invention, optionally in combination with other known ~ntifilng~l agents, according to requirements. Compositions cont:~ining N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular interest.
An ninth aspect of the invention provides a unit dosage form comprising one or more N-alkyl-N-glycosyl derivative of formula l(a) or salts thereof and a physiologically acceptable carrier form~ te~1 for ph~rm~ceutical or V~ y use. The unit dosage form may be formlll~t~l from one or more compounds according to the invention optionally in g :.;~,.
, 56~PLSPE~PCTAMI
combination with other known antifungal agents. By analogy with the previous aspects of the invention unit dosage forms cont~ining N-methyl-N-glycosyl derivatives of formula I (b) are of particular interest.
As mentioned above the nature of the unit dosage form will depend upon the mode 5 of administration. Typically tablets and capsules are suitable for oral administration; crearns and patches are suitable for topical administration with pessaries being suitable for rectal and vaginal administration.
A tenth aspect of the invention provides the use of N-alkyl-N-glycosyl derivative of formula I (a) or salts thereof for the plepdldlion of a medicament for use in the treatment 10 of fungal infections. Compounds wherein the N-alkyl and alkyl ester substituents are both methyl groups are of particular interest.
Structural determinations carried out on the compounds of the invention using spectroscopic methods indicate that the integrity of the parent antibiotic is preserved during the reaction.
The invented process for ~repdldlion of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group gives the desired products without changes in structure of the parent antibiotic. Structure of the obtained compounds was proved using spectroscopic methods. The proof is illustrated by ~iet~-~nin~tion of the structure of N-methyl-N-D-fructosylarnphotericin B methyl ester, of formula 3.
/~CH3 ~ ~ H3 CN ~OH
J~CH3 OH OH OH OH o~CoocH3 OH
H3CJ~O ~OH
OH
= Forrnula 3 ~ d;~ "
1: '137'~56\PLSPECPCTAMI
Electronic spectrum of N-methyl-N-D-fructosylamphotericin B methyl ester is identical with that of the parent antibiotic, that means amphotericin B, and demonstrates that the invented method does not lead to degradation of the polyene chromophore, and a high value of e~tinction (El%lCm = 1300 at 382 nm) confirms ahigh purity ofthe obtained 5 product. Absorption infrared spectrum of the N-methyl-N-D-fructosylamphotericin B
methyl ester demonstrates the band related to stretching vibrations of the ester carbonyl group at 1730 cm~l, and lack of band of free carboxylic group, what means that the carboxylic group was completely transformed into methyl ester group. Complete information on structure of the N-methyl-N-D-fructosylamphotericin B methyl ester was 10 provided by nuclear magnetic resonance spectra (NMR) upon I H (DQF-COSY, ROESY), 13C (DEPT) and heterocorrelated spectra (Varian 300 MHZ spectrometer) allowed anassignment of the formula 3 for the compound.
The most significant 1 H and 13C information are listed in Tables 1 and 2, respectively. The NMR data for aglycone of amphotericin B are in full agreement with literature data - Magn. Reson. Chem.30,275, (1992), P. Sowiriski, J. Pawlak, E. Borowski, P. Gariboldi. 1 H chemical shifts (in DMSO/MeOD solvent system) of N-CH3 (o = 2.35 ppm) and H-1" (2.30 and 3.15 ppm) are characteristic for influence of an amino substituent. After acidification o changes to 2.92 ppm for N-CH3, and to 3.64 for H-1 " due to the protonation of the amino group (o for H-3' changes to 3.19 ppm). These data are 20 supported also by ROE effects between protons NCH3/H3', NCH3/H2', NCH3/Hl"b and I "a/H3'. Coupling constants and ROE indicate 4C 1 conformation of the mycosamine moiety, as it was found before for free arnphotericin B.
Table 1 ~l~S~ chemical shifts lH and ROE effects ofthe ~ crh~nde fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester.
1:'137~.56\PLSPECPCTAMI
Table 1 proton o[ppm] ROE for protons O[ppm] ROE for protons pyridine-d5 methanol-d49: I nMSO-d~: methanol-d., 4: 6 1' 4.77 2', 3', 5', 18b, COOMe 4.18 3', 5' 2' 4.41 1', 3', NMe, 17, COOMe, l"a? 4.04 3',NMe, COOMe
3' 2.0~ l', 2', 5', NMe, l"a, COOMe 1.89 1', 7', NMe, I"b ~' ~.3 8 6' 3 .80 6' 5' 3.61 1',3',6' 3.~5 1',3'.6' 6' 1.~3 4', 5' 1.27 4', 5' l"a 2.56 3',3" (2') 2.30 I"b 3.58 NMe 3.15 3",3' 3" 4.41 I"a? 3.65 I"b
4" ~.76 3.97
5" 4.41 3.63
6"a 4.21 3.57 6"b 4.36 3.76 NMe 2.29 2', 3', l"b, COOMe 2.35 2', 3', l"a, l"b, COOMe COOMe 3.70 1', 2', 3', NMe, 16 3.77 2', NMe ,~"0~) S~
.. ~.~ , r'l37'~56\1'LSPEC'.r'CT~.~,11 Table 2 presents 1 3C-NMR chemical shifts of the disaccharide fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester and their comparison with data for D-fructose.
Table 2 data for fructoses*
~-D-fructopyranose a-D-furanose ~-D-fructofuranose carbon ¦ o [ppm] o [ppm] O [ppm] O [ppm]
1' 98.2 2' 72.4 3' 66.4 4' 69.8 5' 72.3 6' 18.2 I " 62.264. 1 62. 1 63 .9 2" 98. 199. 1 1 05.3 1 02.4 3" 66.970.5 83.0 76.5 ~" 7 1 .2 68.4 77.0 75.5 5" 72.270.0 82.2 8 1 .5 6" 64.664.7 62. 1 63 .3 NMe 40.9 COOMe 5 1.6 * S. N. Rosenthal & J.H. Fendler, Progr. Phys. Org. Chem. 13, 280 (1976).
Comparison of the 13C-NMR data from Table 2 for the fructosyl fragment with literature data for D-fructose indicates the pyranoside form of the sugar substituent. The observed coupling co,.~l~,l~, plese"L~d in Table 3, evidence a boat conformation of the fructopyranoside ring presented by formula 4. Only this conformation of the ring is in full 5 agreement with the measured coupling co~
Table 3 presents coupling c.,"xlu. ~l~ JH H for protons of the ~ cçh~ride fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester (pyridine-ds: methanol-d4 9: 1), 6\PI.SPEC~PCI'A~
coupling constants and chemical shifts of closely coupled spin system H3" - H6" were refined iteratively by computer simulation.
Table 3 protons J [Hz] protons J [Hz]
1',2' -0 l"a, l"b 11.6 2'. 3' 7.2 3",4" 1.05 3~ 9.5 4",5" 8.09 4', 5' 9.2 5", 6"a 5.76 5', 6' 6.0 5", 6"b 3.61 6"a, 6"b -10.86 4", 6"a -0.1 10 The presented results confirm the conformation of the fructosyl fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester presented by formula 4. Other N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group were characterized by methods similar to those described above.
OH
H~
OH
Formula 4 The invention will now be described by reference to the following non-limiting examples. Further embodiments falling wit'nin the scope of the invention will be apparent to a skilled person.
Anti-fungal Activity For all of the prepared compounds ~ntifimg~l activity and also toxicity in vitro were 20 ~ t~rminP~l For the compound with the best properties, that means for the N-methyl-N-D-fructosylamphotericin B methyl ester aspartate, activity and toxicity in vivo were also 1 ' I 37'.4561P~SPEC\I'CTAM I
1 0 determined.
In Vitro Antifungal Activity The antifurlgal activity of compounds was determined following the standard for polyene macrolides procedure. The liquid Sabouraud medium was inoculated with 10~ cells/ml of test organism Candida albicans ATCC 262778 and incubated for 24 hours at 30~C with the tested antibiotic (serial dilutions). Amphotericin B was used as the 5 reference. Compounds were dissolved in DMF and suitable amounts of the solutions were added to the medium. Turbidimetric method (660 nm) was applied to determine the degree of growth inhibition. The concentration of antibiotic at which the growth of fungi was inhibited by 50% was deterrnined from the dose response curve. The obtained ICso value characterized antifungal activity of the compound.
10 In VitroToxicity Toxicity of compounds in vitro against animal cells was determined using standard for polyene macrolides procedure, by the det~rmin~tion of the degree of haemolysis of human erythrocytes. Human erythrocytes isolated from fresh, citrated human blood were washed twice with cold saline. The cells were diluted 250 times with saline and 15 equilibrated for 30 minutes at 37~C. Samples of erythrocytes were incubated with various concentrations of antibiotics (the base solution in DMF) for 30 minlltes at 37~C. After centrifugation the Iysis of erythrocytes was ~ce~ed by ~ . ",i,~tion of the haemoglobin released to the solution. Optical density of the sup~",,.l~.l was measured at 550 nm. The results were ~ sed by EHso value, as concentration of antibiotic at which 50% of20 hemolysis occurred. The values of EHso were read from a curve relating the degree of hemolysis with antibiotic dose.
In vivo Toxicity In vivo toxicity was tletermined for methyl ester of N-methyl-N-D-fructosylamphotericin B L-~L,a,ldle as the m~ximtlm tolerated dose (MTD), and as the 25 acute toxicity (LDso). To determine MTD dose the compound was dissolved in 5%solution of glucose, and ~tlmini~red illLIdvenously and hlL.d~,.;lonealy to Balb/c mice in single and multiple doses. The m~cimllm single tolerated dose was 100 mg/kg for the intravenousandmorethan200mg/kgforthei"lLd~ o.~ lmini~tinn Them;1xi",ll", multiple tolerated dose for illLld~ P~ I - n i ~ l ;on of 100 mg/kg for 5 days was much U .;- s ~
r ~I37\ 5(7~PLSPEC~PCT~
higher. For such dose, to~ic effects were not Qbserved during 20 days of observation.
The acute toxicity, LDso of methyl ester of N-methyl-N-D-fructosylamphotericin BL-~spartate was determined for Swiss Webster female mice of average weight of ~) g.
Various doses of the tested compound, and for comparison, amphotericin B in form of 5 Fungizone, dissolved in 5% glucose were given intravenously to the animals. The ~minictered volume of the solution was 0.5 ml. 0.5 ml of 5 % solution of glucose was ~ iminictered to mice as a control. Every dose of both ~ ~dLions was administered to 5 mice. The animals were observed for 7 days. Next, the animals were killed, and some serum indexes were determined. No increased level of aspartate aminotransferase or 10 creatinine were found in comparison with the control. For methyl ester of N-methyl-N-D-fructosylamphotericin B L-aspartate, the LDso was found to be 400 mg/kg, while for amphotericin B in form of Fungizone it was 6 mg/kg.
Chemotherapeutic Efficacy Chemotherapeutic efficacy of methyl ester of N-methyl-N-D-fructosylamphotericin 15 B L-aspartate was fit~tt~rrnin~?~ using systemic murine candidose model. Candida albicans was grown overnight in Sabouraud dextrose broth at room temperature. The fungal cells were centrifuged, washed twice with 0.9 % solution of sodium chloride, and suspended in physiological salt solution. Female Swiss Webster mice of 25 g weight, were injected intravenously with 105 cells of Candida in 0.2 ml of 0.9 % sodium chloride solution.
20 Initially, the infection was systemic, but by 2 to 3 days it was localized to the kidneys.
Untreated animals usually died between 7 to 14 days post-infection. Three days post-infection, animals were treated intravenously, twice a day, for 5 consecutive days with a S to 6 hours interval. Preparation was :~lmini~tered as a solution in 5~/O glucose. The arumals were observed for 5 weeks starting from the day of infection. After this time, the 25 surviving animals were sacrificed, their kidneys were removed, homogenized in sterile water, and the homogenate was plated on Sabouraud dextrose agar, and the grown colonies of Candida were counted. The chemothc.~ Lic effectiveness was represented as a dose of mglkg, which in the above test resulted in a survival of 50 % of ~nim~l~, and on clearance of Candida from kidneys of half of the mice. The dose, called EDso, was calculated using 30 a method given in J. Hyg. 27, 493, (1938). The values of EDso for methyl ester of N-methyl-N-D-fructosylamphotericin B L-aspartate are 2.3 mg/kg based on the survival, D :~
r \137'~56~PLSrEC~PCT.
and 6 mg/kg based on the kidneys clearance.
Preparative Examples N-Methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group. their salts, and methods of preparation are illustrated by the examples given below.
5 Example r.
I g of amphotericin B (E1%1cm = 1350 at 382 nm, MeOH) was dissolved in 15 ml of N,N-dimethylformamide, 0.3 g of D-glucose was added, and the mi~ture was stirred in l~rkne~ at 37~C for 40 hours. Next the reaction was cooled and a solid was precipitated with an excess of diethyl ether. The solid was centrifuged, washed twice with diethyl ether 10 and dried under reduced pressure. To remove an excess of glucose the solid was suspended in 20 ml of water, centrifuged, washed twice with small amount of water, twice with acetone, and next twice with diethyl ether. The product was dried under reduced pressure to give 0.98 g of N-D-fructosylamphotericin B (El%1Cm = 1200 at 382 nm, MeOH). The product was dissolved, with stirring, in 10 ml of N,N-dimethylformamide and 50 ml of 15 diethyl ether was added to the solution, to give a fine suspension. The suspension was cooled in ice to 0 - 2~C, and freshly prepared diethyl ether solution of 2.5 mole of diazomethane per 1 mole of N-D-fructosylamphotericin B, was added with vigorous stirring. The reaction was followed by thin layer chromatography on silica gel in chloroform - methanol - water 10:6: 1 v/v solvent system. After completion of the reaction, 20 what took about 2 hours, the excess of diazomethane and diethyl ether was evaporated under reduced ~les~ e at temperature not higher than 40~C. The crude product was precipitated from the residue with an excess of diethyl ether, centrifuged, washed twice with diethyl ether, next with n-hexane, and dried under reduced pressure to give 0.95 g of the crude product. Pure N-methyl-N-D-fructosylamphotericin B methyl ester was isolated from the 25 crude product by column chromatography on Merck Silicagel 60, 70 -230 mesh inchloroform - methanol - water 20: 8: 1 v/v solvent system. Thus, 0.95 g of the crude product was suspended in the mixture of solvents specified above, and if the product dissolved with difficulties, proportions of the same solvents were changed to 10: 6: 1. The undissolved part was centrifuged off, and the sup~lln~ charged on chromatography30 column, next developed in the solvent mixture listed above, but in proportion 20: 8: l v/v.
- ~ -r 13î\~56~PLSPEC\PCTA~
The eluate was analysed on silica plates using chloroform - methanol - water 10: 6: 1 v/v solvent system. The plates were visualized with cerium sulphate reagent. Fractions of Rf = 0.5 - 0.54 cont:~ining pure N-methyl-N-D-fructosyl derivative of amphotericin B
methyl ester were collected. The combined fractions were evaporated under reduced 5 pressure. The dry residue was dissolved in small amount of N,N-dimethylformamide, and the product was precipitated with an excess of diethyl ether, the solid was centrifuged, washed twice with diethyl ether and dried in a vacuum desiccator. 0.137 g of N-methyl-N-D-fructosylamphotericin B methyl ester, E1%1Cm = 1300 on 382 nm in methanol, wasobtained. The proof of structure was given above in the descriptive part. Antifungal 10 activity of the compound against Candida albicans, determined as it was described above, gave ICso = 0.12 ~lg/ml, and toxicity for human erythrocytes, determined as described above, gave EHso value higher than 350 llg/ml. For comparison, EHs0 value for the starting amphotericin B was 1.5 llg/ml. An exact value of EHso for N-methyl-N-D-fructosylamphotericin B methyl ester could not be ~letermined, as above 350 ~g/ml the 15 compound was insoluble under conditions of the experiment.
Example II.
0.5 g of candidin (El%lCm = 1175 at 382 nm, MeOH) and 0.15 g of D-glucose were dissolved in 10 ml of N,N-dimethylformamide and stirred at 37~C for 36 hours. Further procedure was analogous to this of the Example I, and resulted in 0.43 g of 20 N-D-fructosylç~n~ lin, El%1Cm = 1100 at 382 nm in MeOH. The product was methylated with diazometh~ne in diethyl ether, analogously as in the Example I to give 0.4 g of crude product. Pure N-methyl-N-D-fructosylcandidin methyl ester was isolated by columnchromatography, by method similar to that given in the Example I. Fractions cont~ining pure derivative were char~teri7~ on thin layer chromatography by Rf = 0.49 - 0.52. The 25 fractions were combined, evaporated to dryness, dissolved in small amount of N,N-dimethylformamide, and solid was precipitated with diethyl ether. The solid was centrifuged, washed with diethyl ether and dried in vacuum desiccator to give 0.05 g of N-methyl-N-D-fructosylc~n~ 1in methyl ester; El%lCm = 1200 at 382 nm, in MeOH, ICso = 0 75 ~lg/ml and EHs0 above 30011g/ml. Structure of the compound was ~~termined 30 by the same methods as for of N-methyl-N-D-fructosylamphotericin B methyl ester.
~ S'~
r ',137\~56\1'LSPECI'CTA:~I
Example III.
2 g of nystatin (El /~1Cm = 870 at 304 nm, in MeOH) and 0.6 g of D-glucos~ were dissolved in 35 ml of N,N-dimethylacetamide and stirred at 37~C for 40 hours. ~fter completion of the reaction the crude product was precipitated with diethyl ether, 5 centrifuged, and dried under vacuum. Then, an excess of glucose was washed off with small amount of water - acetone 1: 1 mixture, next with acetone, diethyl ether, and the product was centrifuged and dried in vacuum desiccator to give 1.2 g of N-D-fructosylnystatin (El%lCm = 720 at 304 nm, in MeOH). The product was methylated using diazomethane as in the Example I to give 1.25 g of crude product. The product was 10 purified on silica gel column as in the Example I. Fractions cont:~ining pure N-methyl-N-D-fructosylnystatin methyl ester were characterized on thin layer chromatography by Rf=
0.49 - 0.52 as in the Example I. The fractions were combined, evaporated to dryness at 30~C under reduced pressure, dissolved in small amount of N,N-dimethylformamide, and product was precipitated with diethyl ether. The product was centrifuged, washed with 15 diethyl ether and dried in vacuum desiccator to give 0.17 g of N-methyl-~D-fructosylnystatin methyl ester; El%lCm = 900 at 304 nm, in MeOH. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester. For the obtained compound ICso = 6.8 ~Lg/ml and EHs0 above 300 ~g/ml were found.
20 Example IV.
0.79 g of vacidin, the main component of antibiotic complex aureofacin, (E 1 % l cm = 900 at 378 nm, in MeOH) and 0.22 g of D-glucose in 15 ml of N,N-dimethyl~cet~ le were stirred at 37~C for 18 hours. After completion of the reaction the mixture was cooled and solid was precipitated with an excess of diethyl ether. The solid was centrifuged, washed 25 with diethyl ether and dried under reduced ~les~ to give 0.8 g of crude N-D-fructosylvacidin (El%lCm = 720 at 378 nm, in MeOH). The obtained derivative was methylated using diazomethane, as in the Example I to give 0.5 g of crude product. Pure N-methyl-N-D-fructosylvacidin methyl ester was isolated by chromatography on silica gel column similarly as it was described in the Example I, but the column was developed with 30 chloroform - methanol - water 30: 8: 1 solvent system. Fractions cont~ining pure F'137' 156'1'1_5PECI'CTA!~,SI
derivative of vacidin and having on thin layer chromatography value of Rf = 0.53 - 0.55 in chloroform - methanol - water 13: 8: I solvent system were c~llected and combined.
Further procedure was as in the Example III. 0.07 g of N-methyl-N-D-fructosylvacidin methyl estl r was obtained (E I % l cm = 900 at 378 nm, in MeOH) . The compound exhibited ICso = 0 01 llg/ml and EHs0 =: 170 llg/ml. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example V.
0.79 g of candicidin D, the main component of antibiotic complex candicidin. was treated identically as described in the Example IV. 0.1 g of N-methyl-N-D-fructosylcandicidin D
methyl ester was obtained; E l%lcm = 920 at 378 nm, in MeOH, Rf of the compound in thin layer chromatography, under conditions described in the Exarnple IV, was 0.50 - 0.53.
The compound exhibited ICso = 0.01 ,ug/ml and EHs0 = 180 ~g/ml. Structure of thecompound was deterrnined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example VI.
0 79 g of the main component of antibiotic complex trychomycin was treated i-l~n~ic~lly as it was described in the Example IV. 0.1 g of N-methyl-N-fructosyltrychomycin methyl esterwasobtained,El%lcm= 910at378nm,inMeOH'Rfofthecompoundinthinlayer chromatography, under conditions described in the Example IV, was 0.49 - 0.52. The compound exhibited ICso = 0.013 ~g/ml and EHs0 = 165 llg/ml. Structure of the compound was ~l~?te~rnint~d by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example VII.
0.5 g of amphotericin B (E1%1Cm = 1350 at 382 nm, MeOH) and 0.15 g of L-glucose in 8 ml of N,N-dimethylro,.. ~.. icle was stirred at 37~C for 40 hours. Subsequent operations were as in the Example I. 0.065 g of N-methyl-N-L-fructosylamphotericin B
methyl ester was obtained; E1%1Cm = 1280 at 382 nrn, in MeOH. The compound exhibited ICso = 0.42 ~Lg/ml and EHs0 = above 200 ~lg/ml. Structure of the compound -20- ~ ~
F~137~J56\PI,SPEC'I'CTAIMl was determined by a method _ described for methyl ester of N-methyl-N-D-fructosylamphotericin B.
Example VIII.
0.52 g of amphotericin B (E 1%1 cm = 1350 at 382 nm, MeOH) and 0.153 g of D-mannose was dissolved in 10 ml of N,N-dimethylformamide and stirred at 37~C for 40 hours.
Subsequent operations were as in the Example I. Pure derivative was isolated by silica gel column chromatography, in a manner analogous to that of the Example I, fractions having in thin layer chromatography, run according to the method described in the Example I, value of Rf= 0.53 - 0.55 were collected. Subsequent operations were as in the Example I.
0.08 g of N-methyl-N-D-fructosylamphotericin B methyl ester was obtained;
E 1 % l cm = 1280 at 382 nm, in MeOH. The compound exhibited ICs0 = 0.42 ~Lg/ml and EHso above 200 ~Lg/ml. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example IX.
0.5 g of amphotericin B (E1%1Cm = 1350 at 382 nm, MeOH) and 0.3 g of D-lactose in 12 ml of N,N-dimethylformamide was stirred at 37~C for two days. Subsequent operations were as in the Example I. Pure derivative was isolated by silica gel column chromatography, in a manner analogous to that of the Example I, fractions having in thin layer chromatography, run according to the method described in the Example I, value of Rf= 0.25 - 0.30 were collected. 0.08 g of N-methyl-N-D-fructosyl-galactosylamphotericin B methyl ester was obtained; E1%1Cm = 1000 at 382 nm, in MeOH. The compound exhibited ICso = 6.0 ,ug/ml and EHs0 above 200 ,ug/ml.
Example X.
0.5 g of N-methyl-N-D-fructosylamphotericin B methyl ester, prepared according to the Example I, was suspended in 10 ml of water and 0.059 g of aspartic acid dissolved in 2 ml of water was added. The solution of acid was added dropwise, with stirring to effect the solution. The solution was filtered to remove a small amount of residual solid and an excess of acetone was added to the clear filtrate until the whole salt was precipitated. The solid P \ 137\156\PLSPI~C PCTAM I
was filtered off or centrifuged, washed twice with acetone, twice with diethyl ether and dried under reduced pressure, 0.5 g of methyl ester of N-methyl-N-D-fructosylamphotericin B L -aspartate, E I % 1 cm = 11 00 at 3 82 nm, in MeOH, was obtained. Th~in layer chromatography under conditions as in the Example I gave Rf = 0.5 - 0.5~. Product was 5 soluble in N,N-dimethylformamide, dimethylsulphoxide and 5% water solution of glucose.
It was very well soluble in water. The compound exhibited ICso = 0.125 ~Lg/ml and EHso above 350 llg/ml.
~ , ~
.. ~.~ , r'l37'~56\1'LSPEC'.r'CT~.~,11 Table 2 presents 1 3C-NMR chemical shifts of the disaccharide fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester and their comparison with data for D-fructose.
Table 2 data for fructoses*
~-D-fructopyranose a-D-furanose ~-D-fructofuranose carbon ¦ o [ppm] o [ppm] O [ppm] O [ppm]
1' 98.2 2' 72.4 3' 66.4 4' 69.8 5' 72.3 6' 18.2 I " 62.264. 1 62. 1 63 .9 2" 98. 199. 1 1 05.3 1 02.4 3" 66.970.5 83.0 76.5 ~" 7 1 .2 68.4 77.0 75.5 5" 72.270.0 82.2 8 1 .5 6" 64.664.7 62. 1 63 .3 NMe 40.9 COOMe 5 1.6 * S. N. Rosenthal & J.H. Fendler, Progr. Phys. Org. Chem. 13, 280 (1976).
Comparison of the 13C-NMR data from Table 2 for the fructosyl fragment with literature data for D-fructose indicates the pyranoside form of the sugar substituent. The observed coupling co,.~l~,l~, plese"L~d in Table 3, evidence a boat conformation of the fructopyranoside ring presented by formula 4. Only this conformation of the ring is in full 5 agreement with the measured coupling co~
Table 3 presents coupling c.,"xlu. ~l~ JH H for protons of the ~ cçh~ride fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester (pyridine-ds: methanol-d4 9: 1), 6\PI.SPEC~PCI'A~
coupling constants and chemical shifts of closely coupled spin system H3" - H6" were refined iteratively by computer simulation.
Table 3 protons J [Hz] protons J [Hz]
1',2' -0 l"a, l"b 11.6 2'. 3' 7.2 3",4" 1.05 3~ 9.5 4",5" 8.09 4', 5' 9.2 5", 6"a 5.76 5', 6' 6.0 5", 6"b 3.61 6"a, 6"b -10.86 4", 6"a -0.1 10 The presented results confirm the conformation of the fructosyl fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester presented by formula 4. Other N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group were characterized by methods similar to those described above.
OH
H~
OH
Formula 4 The invention will now be described by reference to the following non-limiting examples. Further embodiments falling wit'nin the scope of the invention will be apparent to a skilled person.
Anti-fungal Activity For all of the prepared compounds ~ntifimg~l activity and also toxicity in vitro were 20 ~ t~rminP~l For the compound with the best properties, that means for the N-methyl-N-D-fructosylamphotericin B methyl ester aspartate, activity and toxicity in vivo were also 1 ' I 37'.4561P~SPEC\I'CTAM I
1 0 determined.
In Vitro Antifungal Activity The antifurlgal activity of compounds was determined following the standard for polyene macrolides procedure. The liquid Sabouraud medium was inoculated with 10~ cells/ml of test organism Candida albicans ATCC 262778 and incubated for 24 hours at 30~C with the tested antibiotic (serial dilutions). Amphotericin B was used as the 5 reference. Compounds were dissolved in DMF and suitable amounts of the solutions were added to the medium. Turbidimetric method (660 nm) was applied to determine the degree of growth inhibition. The concentration of antibiotic at which the growth of fungi was inhibited by 50% was deterrnined from the dose response curve. The obtained ICso value characterized antifungal activity of the compound.
10 In VitroToxicity Toxicity of compounds in vitro against animal cells was determined using standard for polyene macrolides procedure, by the det~rmin~tion of the degree of haemolysis of human erythrocytes. Human erythrocytes isolated from fresh, citrated human blood were washed twice with cold saline. The cells were diluted 250 times with saline and 15 equilibrated for 30 minutes at 37~C. Samples of erythrocytes were incubated with various concentrations of antibiotics (the base solution in DMF) for 30 minlltes at 37~C. After centrifugation the Iysis of erythrocytes was ~ce~ed by ~ . ",i,~tion of the haemoglobin released to the solution. Optical density of the sup~",,.l~.l was measured at 550 nm. The results were ~ sed by EHso value, as concentration of antibiotic at which 50% of20 hemolysis occurred. The values of EHso were read from a curve relating the degree of hemolysis with antibiotic dose.
In vivo Toxicity In vivo toxicity was tletermined for methyl ester of N-methyl-N-D-fructosylamphotericin B L-~L,a,ldle as the m~ximtlm tolerated dose (MTD), and as the 25 acute toxicity (LDso). To determine MTD dose the compound was dissolved in 5%solution of glucose, and ~tlmini~red illLIdvenously and hlL.d~,.;lonealy to Balb/c mice in single and multiple doses. The m~cimllm single tolerated dose was 100 mg/kg for the intravenousandmorethan200mg/kgforthei"lLd~ o.~ lmini~tinn Them;1xi",ll", multiple tolerated dose for illLld~ P~ I - n i ~ l ;on of 100 mg/kg for 5 days was much U .;- s ~
r ~I37\ 5(7~PLSPEC~PCT~
higher. For such dose, to~ic effects were not Qbserved during 20 days of observation.
The acute toxicity, LDso of methyl ester of N-methyl-N-D-fructosylamphotericin BL-~spartate was determined for Swiss Webster female mice of average weight of ~) g.
Various doses of the tested compound, and for comparison, amphotericin B in form of 5 Fungizone, dissolved in 5% glucose were given intravenously to the animals. The ~minictered volume of the solution was 0.5 ml. 0.5 ml of 5 % solution of glucose was ~ iminictered to mice as a control. Every dose of both ~ ~dLions was administered to 5 mice. The animals were observed for 7 days. Next, the animals were killed, and some serum indexes were determined. No increased level of aspartate aminotransferase or 10 creatinine were found in comparison with the control. For methyl ester of N-methyl-N-D-fructosylamphotericin B L-aspartate, the LDso was found to be 400 mg/kg, while for amphotericin B in form of Fungizone it was 6 mg/kg.
Chemotherapeutic Efficacy Chemotherapeutic efficacy of methyl ester of N-methyl-N-D-fructosylamphotericin 15 B L-aspartate was fit~tt~rrnin~?~ using systemic murine candidose model. Candida albicans was grown overnight in Sabouraud dextrose broth at room temperature. The fungal cells were centrifuged, washed twice with 0.9 % solution of sodium chloride, and suspended in physiological salt solution. Female Swiss Webster mice of 25 g weight, were injected intravenously with 105 cells of Candida in 0.2 ml of 0.9 % sodium chloride solution.
20 Initially, the infection was systemic, but by 2 to 3 days it was localized to the kidneys.
Untreated animals usually died between 7 to 14 days post-infection. Three days post-infection, animals were treated intravenously, twice a day, for 5 consecutive days with a S to 6 hours interval. Preparation was :~lmini~tered as a solution in 5~/O glucose. The arumals were observed for 5 weeks starting from the day of infection. After this time, the 25 surviving animals were sacrificed, their kidneys were removed, homogenized in sterile water, and the homogenate was plated on Sabouraud dextrose agar, and the grown colonies of Candida were counted. The chemothc.~ Lic effectiveness was represented as a dose of mglkg, which in the above test resulted in a survival of 50 % of ~nim~l~, and on clearance of Candida from kidneys of half of the mice. The dose, called EDso, was calculated using 30 a method given in J. Hyg. 27, 493, (1938). The values of EDso for methyl ester of N-methyl-N-D-fructosylamphotericin B L-aspartate are 2.3 mg/kg based on the survival, D :~
r \137'~56~PLSrEC~PCT.
and 6 mg/kg based on the kidneys clearance.
Preparative Examples N-Methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group. their salts, and methods of preparation are illustrated by the examples given below.
5 Example r.
I g of amphotericin B (E1%1cm = 1350 at 382 nm, MeOH) was dissolved in 15 ml of N,N-dimethylformamide, 0.3 g of D-glucose was added, and the mi~ture was stirred in l~rkne~ at 37~C for 40 hours. Next the reaction was cooled and a solid was precipitated with an excess of diethyl ether. The solid was centrifuged, washed twice with diethyl ether 10 and dried under reduced pressure. To remove an excess of glucose the solid was suspended in 20 ml of water, centrifuged, washed twice with small amount of water, twice with acetone, and next twice with diethyl ether. The product was dried under reduced pressure to give 0.98 g of N-D-fructosylamphotericin B (El%1Cm = 1200 at 382 nm, MeOH). The product was dissolved, with stirring, in 10 ml of N,N-dimethylformamide and 50 ml of 15 diethyl ether was added to the solution, to give a fine suspension. The suspension was cooled in ice to 0 - 2~C, and freshly prepared diethyl ether solution of 2.5 mole of diazomethane per 1 mole of N-D-fructosylamphotericin B, was added with vigorous stirring. The reaction was followed by thin layer chromatography on silica gel in chloroform - methanol - water 10:6: 1 v/v solvent system. After completion of the reaction, 20 what took about 2 hours, the excess of diazomethane and diethyl ether was evaporated under reduced ~les~ e at temperature not higher than 40~C. The crude product was precipitated from the residue with an excess of diethyl ether, centrifuged, washed twice with diethyl ether, next with n-hexane, and dried under reduced pressure to give 0.95 g of the crude product. Pure N-methyl-N-D-fructosylamphotericin B methyl ester was isolated from the 25 crude product by column chromatography on Merck Silicagel 60, 70 -230 mesh inchloroform - methanol - water 20: 8: 1 v/v solvent system. Thus, 0.95 g of the crude product was suspended in the mixture of solvents specified above, and if the product dissolved with difficulties, proportions of the same solvents were changed to 10: 6: 1. The undissolved part was centrifuged off, and the sup~lln~ charged on chromatography30 column, next developed in the solvent mixture listed above, but in proportion 20: 8: l v/v.
- ~ -r 13î\~56~PLSPEC\PCTA~
The eluate was analysed on silica plates using chloroform - methanol - water 10: 6: 1 v/v solvent system. The plates were visualized with cerium sulphate reagent. Fractions of Rf = 0.5 - 0.54 cont:~ining pure N-methyl-N-D-fructosyl derivative of amphotericin B
methyl ester were collected. The combined fractions were evaporated under reduced 5 pressure. The dry residue was dissolved in small amount of N,N-dimethylformamide, and the product was precipitated with an excess of diethyl ether, the solid was centrifuged, washed twice with diethyl ether and dried in a vacuum desiccator. 0.137 g of N-methyl-N-D-fructosylamphotericin B methyl ester, E1%1Cm = 1300 on 382 nm in methanol, wasobtained. The proof of structure was given above in the descriptive part. Antifungal 10 activity of the compound against Candida albicans, determined as it was described above, gave ICso = 0.12 ~lg/ml, and toxicity for human erythrocytes, determined as described above, gave EHso value higher than 350 llg/ml. For comparison, EHs0 value for the starting amphotericin B was 1.5 llg/ml. An exact value of EHso for N-methyl-N-D-fructosylamphotericin B methyl ester could not be ~letermined, as above 350 ~g/ml the 15 compound was insoluble under conditions of the experiment.
Example II.
0.5 g of candidin (El%lCm = 1175 at 382 nm, MeOH) and 0.15 g of D-glucose were dissolved in 10 ml of N,N-dimethylformamide and stirred at 37~C for 36 hours. Further procedure was analogous to this of the Example I, and resulted in 0.43 g of 20 N-D-fructosylç~n~ lin, El%1Cm = 1100 at 382 nm in MeOH. The product was methylated with diazometh~ne in diethyl ether, analogously as in the Example I to give 0.4 g of crude product. Pure N-methyl-N-D-fructosylcandidin methyl ester was isolated by columnchromatography, by method similar to that given in the Example I. Fractions cont~ining pure derivative were char~teri7~ on thin layer chromatography by Rf = 0.49 - 0.52. The 25 fractions were combined, evaporated to dryness, dissolved in small amount of N,N-dimethylformamide, and solid was precipitated with diethyl ether. The solid was centrifuged, washed with diethyl ether and dried in vacuum desiccator to give 0.05 g of N-methyl-N-D-fructosylc~n~ 1in methyl ester; El%lCm = 1200 at 382 nm, in MeOH, ICso = 0 75 ~lg/ml and EHs0 above 30011g/ml. Structure of the compound was ~~termined 30 by the same methods as for of N-methyl-N-D-fructosylamphotericin B methyl ester.
~ S'~
r ',137\~56\1'LSPECI'CTA:~I
Example III.
2 g of nystatin (El /~1Cm = 870 at 304 nm, in MeOH) and 0.6 g of D-glucos~ were dissolved in 35 ml of N,N-dimethylacetamide and stirred at 37~C for 40 hours. ~fter completion of the reaction the crude product was precipitated with diethyl ether, 5 centrifuged, and dried under vacuum. Then, an excess of glucose was washed off with small amount of water - acetone 1: 1 mixture, next with acetone, diethyl ether, and the product was centrifuged and dried in vacuum desiccator to give 1.2 g of N-D-fructosylnystatin (El%lCm = 720 at 304 nm, in MeOH). The product was methylated using diazomethane as in the Example I to give 1.25 g of crude product. The product was 10 purified on silica gel column as in the Example I. Fractions cont:~ining pure N-methyl-N-D-fructosylnystatin methyl ester were characterized on thin layer chromatography by Rf=
0.49 - 0.52 as in the Example I. The fractions were combined, evaporated to dryness at 30~C under reduced pressure, dissolved in small amount of N,N-dimethylformamide, and product was precipitated with diethyl ether. The product was centrifuged, washed with 15 diethyl ether and dried in vacuum desiccator to give 0.17 g of N-methyl-~D-fructosylnystatin methyl ester; El%lCm = 900 at 304 nm, in MeOH. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester. For the obtained compound ICso = 6.8 ~Lg/ml and EHs0 above 300 ~g/ml were found.
20 Example IV.
0.79 g of vacidin, the main component of antibiotic complex aureofacin, (E 1 % l cm = 900 at 378 nm, in MeOH) and 0.22 g of D-glucose in 15 ml of N,N-dimethyl~cet~ le were stirred at 37~C for 18 hours. After completion of the reaction the mixture was cooled and solid was precipitated with an excess of diethyl ether. The solid was centrifuged, washed 25 with diethyl ether and dried under reduced ~les~ to give 0.8 g of crude N-D-fructosylvacidin (El%lCm = 720 at 378 nm, in MeOH). The obtained derivative was methylated using diazomethane, as in the Example I to give 0.5 g of crude product. Pure N-methyl-N-D-fructosylvacidin methyl ester was isolated by chromatography on silica gel column similarly as it was described in the Example I, but the column was developed with 30 chloroform - methanol - water 30: 8: 1 solvent system. Fractions cont~ining pure F'137' 156'1'1_5PECI'CTA!~,SI
derivative of vacidin and having on thin layer chromatography value of Rf = 0.53 - 0.55 in chloroform - methanol - water 13: 8: I solvent system were c~llected and combined.
Further procedure was as in the Example III. 0.07 g of N-methyl-N-D-fructosylvacidin methyl estl r was obtained (E I % l cm = 900 at 378 nm, in MeOH) . The compound exhibited ICso = 0 01 llg/ml and EHs0 =: 170 llg/ml. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example V.
0.79 g of candicidin D, the main component of antibiotic complex candicidin. was treated identically as described in the Example IV. 0.1 g of N-methyl-N-D-fructosylcandicidin D
methyl ester was obtained; E l%lcm = 920 at 378 nm, in MeOH, Rf of the compound in thin layer chromatography, under conditions described in the Exarnple IV, was 0.50 - 0.53.
The compound exhibited ICso = 0.01 ,ug/ml and EHs0 = 180 ~g/ml. Structure of thecompound was deterrnined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example VI.
0 79 g of the main component of antibiotic complex trychomycin was treated i-l~n~ic~lly as it was described in the Example IV. 0.1 g of N-methyl-N-fructosyltrychomycin methyl esterwasobtained,El%lcm= 910at378nm,inMeOH'Rfofthecompoundinthinlayer chromatography, under conditions described in the Example IV, was 0.49 - 0.52. The compound exhibited ICso = 0.013 ~g/ml and EHs0 = 165 llg/ml. Structure of the compound was ~l~?te~rnint~d by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example VII.
0.5 g of amphotericin B (E1%1Cm = 1350 at 382 nm, MeOH) and 0.15 g of L-glucose in 8 ml of N,N-dimethylro,.. ~.. icle was stirred at 37~C for 40 hours. Subsequent operations were as in the Example I. 0.065 g of N-methyl-N-L-fructosylamphotericin B
methyl ester was obtained; E1%1Cm = 1280 at 382 nrn, in MeOH. The compound exhibited ICso = 0.42 ~Lg/ml and EHs0 = above 200 ~lg/ml. Structure of the compound -20- ~ ~
F~137~J56\PI,SPEC'I'CTAIMl was determined by a method _ described for methyl ester of N-methyl-N-D-fructosylamphotericin B.
Example VIII.
0.52 g of amphotericin B (E 1%1 cm = 1350 at 382 nm, MeOH) and 0.153 g of D-mannose was dissolved in 10 ml of N,N-dimethylformamide and stirred at 37~C for 40 hours.
Subsequent operations were as in the Example I. Pure derivative was isolated by silica gel column chromatography, in a manner analogous to that of the Example I, fractions having in thin layer chromatography, run according to the method described in the Example I, value of Rf= 0.53 - 0.55 were collected. Subsequent operations were as in the Example I.
0.08 g of N-methyl-N-D-fructosylamphotericin B methyl ester was obtained;
E 1 % l cm = 1280 at 382 nm, in MeOH. The compound exhibited ICs0 = 0.42 ~Lg/ml and EHso above 200 ~Lg/ml. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example IX.
0.5 g of amphotericin B (E1%1Cm = 1350 at 382 nm, MeOH) and 0.3 g of D-lactose in 12 ml of N,N-dimethylformamide was stirred at 37~C for two days. Subsequent operations were as in the Example I. Pure derivative was isolated by silica gel column chromatography, in a manner analogous to that of the Example I, fractions having in thin layer chromatography, run according to the method described in the Example I, value of Rf= 0.25 - 0.30 were collected. 0.08 g of N-methyl-N-D-fructosyl-galactosylamphotericin B methyl ester was obtained; E1%1Cm = 1000 at 382 nm, in MeOH. The compound exhibited ICso = 6.0 ,ug/ml and EHs0 above 200 ,ug/ml.
Example X.
0.5 g of N-methyl-N-D-fructosylamphotericin B methyl ester, prepared according to the Example I, was suspended in 10 ml of water and 0.059 g of aspartic acid dissolved in 2 ml of water was added. The solution of acid was added dropwise, with stirring to effect the solution. The solution was filtered to remove a small amount of residual solid and an excess of acetone was added to the clear filtrate until the whole salt was precipitated. The solid P \ 137\156\PLSPI~C PCTAM I
was filtered off or centrifuged, washed twice with acetone, twice with diethyl ether and dried under reduced pressure, 0.5 g of methyl ester of N-methyl-N-D-fructosylamphotericin B L -aspartate, E I % 1 cm = 11 00 at 3 82 nm, in MeOH, was obtained. Th~in layer chromatography under conditions as in the Example I gave Rf = 0.5 - 0.5~. Product was 5 soluble in N,N-dimethylformamide, dimethylsulphoxide and 5% water solution of glucose.
It was very well soluble in water. The compound exhibited ICso = 0.125 ~Lg/ml and EHso above 350 llg/ml.
~ , ~
Claims (43)
1. N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group of general formula 1(a), wherein M represents residue of an antifungal antibiotic of polyene macrolide group, R represents a part of sugar residue formed by reaction of the antibiotic with a mono or oligosaccharide, and each of R1 and R2, which may be the same or different, represents a C 1-4 alkyl group.
Formula 1(a)
Formula 1(a)
2. N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group according to claim 1 wherein M is selected from amphotericin B, candidin, candidoin, candidinin, mycoheptin, nystatin, polyfungin, aureofacin, vacidin, trichomycin or candicidin.
3. N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group according to either claim 1 or 2 wherein the mono or oligosaccharide from which the sugar residue R is derived is selected from D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose.
4. N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group according to any one of claims 1 to 3 wherein R1 and R2 are methyl groups.
5. Salts of N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group of general formula 2(a) wherein M, R, R1 and R2 are defined according to any one of claims 1 to 4 and A represents an anion of an organic or inorganic acid.
Formula 2(a)
Formula 2(a)
6. Salts of N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group according to claim 5 wherein A is the anion of L-aspartic acid.
7. A method of preparation of N-alkyl-N-glycosyl derivatives of alkyl esters of antifungal antibiotics of polyene macrolide group as defined in any one of claims 1 to 4 which comprises the steps of reacting a polyene macrolide antibiotic with a mono or oligosaccharide, the reaction being characterised by the occurrence of a simultaneous Amadori rearrangement, to give the N-glycosyl derivatives of the polyene macrolide antibiotics; isolating the product of the Amadori rearrangement;
treating the product with an alkylating agent and purifying the crude product.
treating the product with an alkylating agent and purifying the crude product.
8. A method of preparation according to claim 7 wherein product of the Amadori rearrangement is isolated in the form of a suspension by precipitation from the solution in which the rearrangement occurs.
9. A method of preparation according to claim 7 or claim 8 wherein N, N-dimethylformamide is used to support the Amadori rearrangement.
10. A method of preparation according to any one of claims 7 to 9 wherein diethyl ether is used to effect formation of a suspension by precipitation of the product of the Amadori rearrangement.
11. A method of preparation according to any one of claims 7 to 10 wherein alkylation of the product of the Amadori rearrangement is carried out at reduced temperature
12. A method of preparation according to claim 10 wherein alkylation is carried out at temperatures of between -5°C and +5°C.
13. A method of preparation according to any one of claims 7 to 12 wherein the alkylating agent is an ethereal solution of diazomethane.
14. A method of preparation of salts of N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics of general formula 2(a) as defined in claim 5 or claim 6 which comprises the steps of suspending an N-alkyl-N-glycosyl derivative prepared according to any one of claims 7 to 13 in sufficient water to effect formation of a homogeneous suspension, acidifying the resulting suspension and isolating the product.
15. A method of preparation of salts of N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to claim 14 wherein organic or inorganicacids are used to acidify the suspension.
16. A method of preparation of salts N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to claim 15 wherein L-aspartic acid is used to acidify the suspension.
17. A method of preparation of salts N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to any one of claims 14 to 16 wherein isolation of the pure product is effected by precipitation of the crude product with an organic solvent followed by subsequent washing with an additional appropriate solvent and drying.
18. A method of preparation of salts N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to any one of claims 14 to 17 wherein the solvent used to precipitate the crude product is miscible with water.
19. A method of preparation of salts N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to claim 18 wherein the solvent is acetone.
20. A method of preparation of salts N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to any one of claims 17 to 19 wherein the additional appropriate solvents employed for washing the product are selected from acetone and diethyl ether.
21. A method of preparation of salts N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics according to any one of claims 14 to 20 wherein the product is dried under reduced pressure.
22. N-alkyl-N-glycosyl derivatives of formula 1(a) or salts thereof according to any one of claims 1 to 6 for use in therapy.
23 A method for the treatment of fungal infections in humans and animals which comprises the administration thereto of N-alkyl-N-glycosyl derivatives of formula 1 (a) or salts thereof according to any one of claims 1 to 6.
24. A method for the treatment of fungal infections in humans and animals which comprises the administration thereto of N-methyl-N-glycosyl derivatives of formula 1 (a) or salts thereof according to any one of claims 1 to 6.
25. A composition for use in the treatment of fungal infections comprising an N-alkyL-N-glycosyl derivative of formula 1 (a) or salts thereof according to any one of claims 1 to 6 and a physiologically acceptable carrier.
26. A unit dosage form comprising one or more N-alkyl-N-glycosyl derivative of formula 1 (a) or salts thereof and a physiologically acceptable carrier formulated for pharmaceutical or veterinary use.
27. A composition for use in the treatment of fungal infections according to claim 25 or a unit dosage form according to claim 26 wherein the N-alkyl-N-glycosyl derivative of formula 1(a) or salts thereof includes N-methyl-N-glycosyl derivatives of themethyl esters of formula 1(b) or the salts thereof.
28. A composition or unit dosage form according to any one of claims 25 to 27 which further comprises a known anti-fungal agent.
29. A composition or unit dosage form according to any one of claims 25 to 28 which is formulated for intravenous, intra peritoneal, oral, topical, subcutaneous, rectal or vaginal administration.
30. Use of N-alkyl-N-glycosyl derivatives of alkyl esters of formula 1 (a) or salts thereof according to any one of claims 1 to 6 for the preparation of a medicament for use in the treatment of fungal infections.
31. Use according to claim 30 wherein the N-alkyl and alkyl ester substituents are both methyl groups.
32. Use of N-alkyl-N-glycosyl derivative of formula 1(a) or salts thereof according to any one of claims 1 to 6 for the treatment of fungal infections.
33. Use according to claim 32 wherein the N-alkyl and alkyl ester substituents are both methyl groups.
34. Use according to claim 32 or claim 33 wherein the fungal infection is external or internal.
35. N-Methyl-N-glycosyl derivatives of methyl esters of antifungal antibiotics of polyene macrolide group presented by general formula 1(b), wherein M represents residue of an antifungal antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide,preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement.
Formula 1(b)
Formula 1(b)
36. Derivatives according to claim 37, wherein the antifungal antibiotic of polyene macrolide group is amphotericin B, or candidin, or candidoin, or candidinin, or mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or candicidin.
37. Salts of N-methyl-N-glycosyl derivatives of antifungal antibiotics of polyene macrolide group presented by general formula 2(b) wherein M represents residue of an antifungal antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide,preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anionof organic or inorganic acid.
Formula 2(b)
Formula 2(b)
38. Salts, according to claim 39, wherein the antifungal antibiotic of polyene macrolide group is amphotericin B, or candidin, or candidoin, or candidinin, or mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or candicidin.
39. Salts according to claims 39 or 40, wherein A relates to the anion of L-aspartic acid.
40. Process for preparation of N-methyl-N-glycosyl derivatives of methyl esters of antifungal antibiotics of polyene macrolide group presented by general formula 1 (b), wherein M represents residue of an antifungal antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivatives of antibiotics of polyene macrolide group are transformed into suspension by precipitation withsolvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5°C
to +5°C, stirred, and isolated by evaporation of solvents, and precipitation from the concentrated solution, preferably by excess of diethyl ether, the crude product is purified according to known procedures.
to +5°C, stirred, and isolated by evaporation of solvents, and precipitation from the concentrated solution, preferably by excess of diethyl ether, the crude product is purified according to known procedures.
41. Process for preparation of salts of N-methyl-N-glycosyl derivatives of methyl esters of antifimgal antibiotics of polyene macrolide group presented by general formula 1(b), wherein M represents residue of an antifungal antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivatives of antibiotics of polyene macrolide group are transformed into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5°C to +5°C, stirred, and isolated by evaporation of solvents, and precipitation from the concentrated solution, preferably by an excess of diethyl ether, the crude product is purified according to known procedures, subsequently, the obtained derivative, as a solid, is suspended in small amount of water, and stoichiometric amount of organic or inorganic acid is added, next the product isprecipitated from the formed solution by an excess of organic solvent miscible with water, preferably acetone, the solid is washed, preferably with acetone and subsequently preferably with diethyl ether, and dried, preferably under reduced pressure.
42. Method for treatment of external and internal fungal infections in humans and animals, wherein N-methyl-N-glycosyl derivatives of methyl esters of antifungal antibiotics of polyene macrolide group presented by general formula 1 (a) 1, wherein M represents residue of an antifungal antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement are used to treat the infections.
43. Method for treatment of external and internal fungal infections in humans and animals, wherein salts of N-methyl-N-glycosyl derivatives of methyl esters of antifungal antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antifungal antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anion of organic or inorganic acid are used to treat the infections.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PLP.308583 | 1995-05-13 | ||
| PL95308583A PL180253B1 (en) | 1995-05-13 | 1995-05-13 | N-methyl n-glycoside derivatives of methyl esters of antibiotic from the group of polyene macrolydes and their salts, method of obtaining them and using them in medical care |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2220771A1 true CA2220771A1 (en) | 1996-11-14 |
Family
ID=20065009
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002220771A Abandoned CA2220771A1 (en) | 1995-05-13 | 1996-05-10 | Antibiotics |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0825995A1 (en) |
| JP (1) | JPH11504647A (en) |
| KR (1) | KR19990014843A (en) |
| AU (1) | AU716883B2 (en) |
| CA (1) | CA2220771A1 (en) |
| IL (1) | IL118233A0 (en) |
| MX (1) | MX9708637A (en) |
| NZ (1) | NZ307592A (en) |
| PL (1) | PL180253B1 (en) |
| WO (1) | WO1996035701A1 (en) |
| ZA (1) | ZA963787B (en) |
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| US5981721A (en) * | 1997-10-23 | 1999-11-09 | Karykion Corporation | Polyene macrolide schiff bases, their alkyl esters and processes for preparing polyene macrolide alkyl ester salts thereof |
| CA2394833A1 (en) | 2000-01-14 | 2001-07-19 | Binh T. Dang | Derivatives of polyene macrolides and preparation and use thereof |
| KR100687748B1 (en) * | 2005-06-23 | 2007-02-27 | 한국전자통신연구원 | Method and System for Fast Handover being independent of Mobile Node |
| GB0712881D0 (en) * | 2007-07-03 | 2007-08-15 | Biosergen As | Compounds |
| IN2014DN07481A (en) | 2012-03-09 | 2015-04-24 | Blirt S A | |
| CA2876074A1 (en) | 2012-06-15 | 2013-12-19 | Blirt S.A. | N-substituted second generation derivatives of antifungal antibiotic amphotericin b and methods of their preparation and application |
| SG11201610162XA (en) | 2014-06-12 | 2017-01-27 | Shionogi & Co | Polyene macrolide derivative |
| US9957290B2 (en) * | 2015-04-15 | 2018-05-01 | Sfunga Therapeutics, Inc. | Derivatives of amphotericin B |
| CN112920238B (en) * | 2021-01-27 | 2022-09-30 | 河南农业大学 | Sugar amine Amadori derivative and preparation method and application thereof |
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| FI60877C (en) * | 1971-08-13 | 1982-04-13 | Politechnika Gdanska | PROTECTION OF THERAPEUTIC ACTIVITIES OF THERAPEUTIC ACTIVITIES WITH A DISPENSABLE POLYENMACROLIDANTIBIOTICS-N-GLYCOSYL DERIVATIVES |
| US4002741A (en) * | 1974-04-24 | 1977-01-11 | Valter Osvaldovich Kulbakh | Meglumine complex fungicidal polyene macrolide antibiotic compositions and treatment method |
| US4195172A (en) * | 1976-04-22 | 1980-03-25 | Politechnika Gdanska | Salts of N-glycosyl derivatives of polyene macrolides, especially N-methylglucamine salts as well as the method of their preparation |
| US4144328A (en) * | 1977-02-28 | 1979-03-13 | Vainshtein Viktor A | N,N,N-Trimethyl derivatives of polyene amphoteric antibiotics, process of producing same and pharmaceutical composition |
| FR2654339B1 (en) * | 1989-11-14 | 1994-10-28 | Mayoly Spindler Laboratoires | NOVEL SOLUBLE AND NON-TOXIC DERIVATIVES OF BASIC POLYENIC MACROLIDES, THEIR PREPARATION AND THEIR APPLICATIONS. |
-
1995
- 1995-05-13 PL PL95308583A patent/PL180253B1/en not_active IP Right Cessation
-
1996
- 1996-05-10 JP JP8533895A patent/JPH11504647A/en active Pending
- 1996-05-10 AU AU56983/96A patent/AU716883B2/en not_active Ceased
- 1996-05-10 KR KR1019970708187A patent/KR19990014843A/en not_active Application Discontinuation
- 1996-05-10 MX MX9708637A patent/MX9708637A/en unknown
- 1996-05-10 EP EP96915094A patent/EP0825995A1/en not_active Withdrawn
- 1996-05-10 CA CA002220771A patent/CA2220771A1/en not_active Abandoned
- 1996-05-10 NZ NZ307592A patent/NZ307592A/en unknown
- 1996-05-10 WO PCT/GB1996/001144 patent/WO1996035701A1/en not_active Application Discontinuation
- 1996-05-13 IL IL11823396A patent/IL118233A0/en unknown
- 1996-05-13 ZA ZA9603787A patent/ZA963787B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IL118233A0 (en) | 1996-09-12 |
| EP0825995A1 (en) | 1998-03-04 |
| KR19990014843A (en) | 1999-02-25 |
| AU5698396A (en) | 1996-11-29 |
| NZ307592A (en) | 1999-01-28 |
| MX9708637A (en) | 1998-02-28 |
| AU716883B2 (en) | 2000-03-09 |
| PL308583A1 (en) | 1996-11-25 |
| JPH11504647A (en) | 1999-04-27 |
| WO1996035701A1 (en) | 1996-11-14 |
| PL180253B1 (en) | 2001-01-31 |
| ZA963787B (en) | 1997-11-13 |
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